Chimeric proteins comprising the extracellular domain of murine Ob receptor

ABSTRACT

The present invention relates to the discovery, identification and characterization of nucleotides that encode Ob receptor (ObR), a receptor protein that participates in mammalian body weight regulation. The invention encompasses obR nucleotides, host cell expression systems, ObR proteins, fusion proteins, polypeptides and peptides, antibodies to the receptor, transgenic animals that express an obR transgene, or recombinant knock-out animals that do not express the ObR, antagonists and agonists of the receptor, and other compounds that modulate obR gene expression or ObR activity that can be used for diagnosis, drug screening, clinical trial monitoring, and/or the treatment of body weight disorders, including but not limited to obesity, cachexia and anorexia.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 08/638,524 filedApr. 26, 1996 (pending), which is a continuation-in-part of applicationSer. No. 08/599,455, filed Jan. 22, 1996 (now U.S. Pat. No. 5,972,621),which is a continuation-in-part of application Ser. No. 08/583,153,filed Dec. 28, 1995 (pending), which is a continuation-in-part ofapplication Ser. No. 08/570,142, filed Dec. 11, 1995 (pending), which isa continuation-in-part of application Ser. No. 08/569,485, filed Dec. 8,1995 (now abandoned), which is a continuation-in-part of applicationSer. No. 08/566,622, filed Dec. 4, 1995 (now abandoned), which is acontinuation-in-part of application Ser. No. 08/562,663, filed Nov. 27,1995 (now abandoned).

1. INTRODUCTION

The present invention relates to the discovery, identification andcharacterization of nucleotides that encode Ob receptor (ObR), areceptor protein that participates in mammalian body weight regulation.The invention encompasses obR nucleotides, host cell expression systems,ObR proteins, fusion proteins, polypeptides and peptides, antibodies tothe receptor, transgenic animals that express an obR transgene, orrecombinant knock-out animals that do not express the ObR, antagonistsand agonists of the receptor, and other compounds that modulate obR geneexpression or ObR activity that can be used for diagnosis, drugscreening, clinical trial monitoring, and/or the treatment of bodyweight disorders, including but not limited to obesity, cachexia andanorexia.

2. BACKGROUND OF THE INVENTION

Obesity represents the most prevalent of body weight disorders, and itis the most important nutritional disorder in the western world, withestimates of its prevalence ranging from 30% to 50% within themiddle-aged population. Other body weight disorders, such as anorexianervosa and bulimia nervosa which together affect approximately 0.2% ofthe female population of the western world, also pose serious healththreats. Further, such disorders as anorexia and cachexia (wasting) arealso prominent features of other diseases such as cancer, cysticfibrosis, and AIDS.

Obesity, defined as an excess of body fat relative to lean body mass,also contributes to other diseases. For example, this disorder isresponsible for increased incidences of diseases such as coronary arterydisease, stroke, and diabetes. (See, e.g., Nishina, P. M. et al., 1994,Metab. 43:554-558. ) Obesity is not merely a behavioral problem, i.e.,the result of voluntary hyperphagia. Rather, the differential bodycomposition observed between obese and normal subjects results fromdifferences in both metabolism and neurologic/metabolic interactions.These differences seem to be, to some extent, due to differences in geneexpression, and/or level of gene products or activity (Friedman, J. M.et al., 1991, Mammalian Gene 1:130-144).

The epidemiology of obesity strongly shows that the disorder exhibitsinherited characteristics (Stunkard, 1990, N. Eng. J. Med. 322:1483).Moll et al. have reported that, in many populations, obesity seems to becontrolled by a few genetic loci (Moll et al. 1991, Am. J. Hum. Gen.49:1243). In addition, human twin studies strongly suggest a substantialgenetic basis in the control of body weight, with estimates ofheritability of 80-90% (Simopoulos, A. P. & Childs B., eds., 1989, in“Genetic Variation and Nutrition in Obesity”, World Review of Nutritionand Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976,Acta. Paediatr. Scand. 65:279-287).

Studies of non-obese persons who deliberately attempted to gain weightby systematically over-eating were found to be more resistant to suchweight gain and able to maintain an elevated weight only by very highcaloric intake. In contrast, spontaneously obese individuals are able tomaintain their status with normal or only moderately elevated caloricintake. In addition, it is a commonplace experience in animal husbandrythat different strains of swine, cattle, etc., have differentpredispositions to obesity. Studies of the genetics of human obesity andof models of animal obesity demonstrate that obesity results fromcomplex defective regulation of both food intake, food induced energyexpenditure and of the balance between lipid and lean body anabolism.

There are a number of genetic diseases in man and other species whichfeature obesity among their more prominent symptoms, along with,frequently, dysmorphic features and mental retardation. For example,Prader-Willi syndrome (PWS) affects approximately 1 in 20,000 livebirths, and involves poor neonatal muscle tone, facial and genitaldeformities, and generally obesity.

In addition to PWS, many other pleiotropic syndromes which includeobesity as a symptom have been characterized. These syndromes are moregenetically straightforward, and appear to involve autosomal recessivealleles. The diseases, which include, among others, Ahlstroem,Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.

A number of models exist for the study of obesity (see, e.g., Bray, G.A., 1992, Prog. Brain Res. 93:333-341, and Bray, G. A., 1989, Amer. J.Clin. Nutr. 5:891-902). For example, animals having mutations which leadto syndromes that include obesity symptoms have been identified, andattempts have been made to utilize such animals as models for the studyof obesity. The best studied animal models, to date, for genetic obesityare mice models. For reviews, see e.g., Friedman, J. M. et al., 1991,Mamm. Gen. 1:130-144; Friedman, J. M. and Liebel, R. L., 1992, Cell69:217-220.)

Studies utilizing mice have confirmed that obesity is a very complextrait with a high degree of heritability. Mutations at a number of locihave been identified which lead to obese phenotypes. These include theautosomal recessive mutations obese (ob), diabetes (db), fat (fat) andtubby (tub). In addition, the autosomal dominant mutations Yellow at theagouti locus and Adipose (Ad) have been shown to contribute to an obesephenotype.

The ob and db mutations are on chromosomes 6 and 4, respectively, butlead to a complex, clinically similar phenotype of obesity, evidentstarting at about one month of age, which includes hyperphagia, severeabnormalities in glucose and insulin metabolism, very poorthermoregulation and non-shivering thermogenesis, and extreme torpor andunderdevelopment of the lean body mass. This complex phenotype has madeit difficult to identify the primary defect attributable to themutations (Bray G. A., et al., 1989 Amer. J. Clin. Nutr. 5:891-902).

Using molecular and classical genetic markers, the db gene has beenmapped to midchromosome 4 (Friedman et al., 1991, Mamm. Gen. 1:130-144).The mutation maps to a region of the mouse genome that is syntonic withhuman, suggesting that, if there is a human homolog of db, it is likelyto map to human chromosome 1p.

The ob gene and its human homologue have recently been cloned (Zhang, Y.et al., 1994, Nature 372:425-432). The gene appears to produce a 4.5 kbadipose tissue messenger RNA which contains a 167 amino acid openreading frame. The predicted amino acid sequence of the ob gene productindicates that it is a secreted protein and may, therefore, play a roleas part of a signalling pathway from adipose tissue which may serve toregulate some aspect of body fat deposition. Further, recent studieshave shown that recombinant Ob protein, also known as leptin, whenexogenously administered, can at least partially correct theobesity-related phenotype exhibited by ob mice (Pelleymounter, M. A. etal., 1995, Science 269:540-543; Halalas, J. L. et al., 1995, Science269:543-546; Campfield, L. A. et al., 1995, Science 269:546-549). Recentstudies have suggested that obese humans and rodents (other than ob/obmice) are not defective in their ability to produce ob mRNA or protein,and generally produce higher levels than lean individuals (Maffei etal., 1995, Nature Med. 1 (11):1155-1161; Considine et al., 1995, J.Clin. Invest. 95(6):2986-2988; Lohnqvist et al., 1995, Nature Med.1:950-953; Hamilton et al., 1995, Nature Med. 1:953-956). These datasuggest that resistance to normal or elevated levels of Ob may be moreimportant than inadequate Ob production in human obesity. However, thereceptor for the ob gene product, thought to be expressed in thehypothalamus, remains elusive.

Homozygous mutations at either the fat or tub loci cause obesity whichdevelops more slowly than that observed in ob and db mice (Coleman, D.L., and Eicher, E. M., 1990, J. Heredity 81:424-427), with tub obesitydeveloping slower than that observed in fat animals. This feature of thetub obese phenotype makes the development of tub obese phenotype closestin resemblance to the manner in which obesity develops in humans. Evenso, however, the obese phenotype within such animals can becharacterized as massive in that animals eventually attain body weightswhich are nearly two times the average weight seen in normal mice.

The fat mutation has been mapped to mouse chromosome 8, while the tubmutation has been mapped to mouse chromosome 7. According to Naggert etal., the fat mutation has recently been identified (Naggert, J. K., etal., 1995, Nature Genetics 10:135-141). Specifically, the fat mutationappears to be a mutation within the Cpe locus, which encodes thecarboxypeptidase (Cpe) E protein. Cpe is an exopeptidase involved in theprocessing of prohormones, including proinsulin.

The dominant Yellow mutation at the agouti locus, causes a pleiotropicsyndrome which causes moderate adult onset obesity, a yellow coat color,and a high incidence of tumor formation (Herberg, L. and Coleman, D. L.,1977, Metabolism 26:59), and an abnormal anatomic distribution of bodyfat (Coleman, D. L., 1978, Diabetologia 14:141-148). This mutation mayrepresent the only known example of a pleiotropic mutation that causesan increase, rather than a decrease, in body size. The mutation causesthe widespread expression of a protein which is normally seen only inneonatal skin (Michaud, E. J. et al., 1994, Genes Devel. 8:1463-1472).

Other animal models include fa/fa (fatty) rats, which bear manysimilarities to the ob/ob and db/db mice, discussed above. Onedifference is that, while fa/fa rats are very sensitive to cold, theircapacity for non-shivering thermogenesis is normal. Torpor seems to playa larger part in the maintenance of obesity in fa/fa rats than in themice mutants. In addition, inbred mouse strains such as NZO mice andJapanese KK mice are moderately obese. Certain hybrid mice, such as theWellesley mouse, become spontaneously fat. Further, several desertrodents, such as the spiny mouse, do not become obese in their naturalhabitats, but do become so when fed on standard laboratory feed.

Animals which have been used as models for obesity have also beendeveloped via physical or pharmacological methods. For example,bilateral lesions in the ventromedial hypothalamus (VMH) andventrolateral hypothalamus (VLH) in the rat are associated,respectively, with hyperphagia and gross obesity and with aphagia,cachexia and anorexia. Further, it has been demonstrated that feedingmonosodium-glutamate (MSG) or gold thioglucose to newborn mice alsoresults in an obesity syndrome.

Each of the rodent obesity models is accompanied by alterations incarbohydrate metabolism resembling those in Type II diabetes in man. Forexample, from both ob and db, congenic C57BL/KS mice develop a severediabetes with ultimate β cell necrosis and islet atrophy, resulting in arelative insulinopenia, while congenic C57BL/6J ob and db mice develop atransient insulin-resistant diabetes that is eventually compensated by βcell hypertrophy resembling human Type II diabetes.

With respect to ob and db mice, the phenotype of these mice resembleshuman obesity in ways other than the development of diabetes, in thatthe mutant mice eat more and expend less energy than do lean controls(as do obese humans). This phenotype is also quite similar to that seenin animals with lesions of the ventromedial hypothalamus, which suggeststhat both mutations may interfere with the ability to properly integrateor respond to nutritional information within the central nervous system.Support for this hypothesis comes from the results of parabiosisexperiments (Coleman, D. L. 1973, Diabetologica 9:294-298) that suggestob mice are deficient in a circulating satiety factor and that db miceare resistant to the effects of the ob factor. These experiments haveled to the conclusion that obesity in these mutant mice may result fromdifferent defects in an afferent loop and/or integrative center of thepostulated feedback mechanism that controls body composition.

In summary, therefore, obesity, which poses a major, worldwide healthproblem, represents a complex, highly heritable trait. Given theseverity, prevalence and potential heterogeneity of such disorders,there exists a great need for the identification of those genes and geneproducts that participate in the control of body weight.

It is an objective of the invention to provide modulators of bodyweight, to provide methods for diagnosis of body weight disorders, toprovide therapy for such disorders and to provide an assay system forthe screening of substances which can be used to control body weight.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification andcharacterization of nucleotides that encode Ob receptor (ObR), a novelreceptor protein that participates in the control of mammalian bodyweight. ObR, described for the first time herein, is a transmembraneprotein that spans the cellular membrane once and is involved in signaltransduction triggered by the binding of its natural ligand, Ob, alsoknown as leptin. ObR has amino acid sequence motifs found in the Class Icytokine receptor family, and is most related to the gp130 signaltransducing component of the IL-6 receptor, the G-CSF receptor, and theLIF receptor. The results presented in the working examples hereindemonstrate that a long-form ObR (predominantly expressed in thehypothalamus) transduces signal via a STAT mediated pathway typical ofIL-6 type cytokine receptors, whereas a major naturally occurringtruncated form or a mutant form found in obese db/db mice does not. Thelong form ObR can mediate activation of STAT proteins and stimulatetranscription through IL-6 responsive gene elements. Reconstitutionexperiments indicate that, although ObR mediates intracellular signalswith a specificity similar to IL-6 type cytokine receptors, signalingappears to be independent of the gp130 signal transducing component ofthe IL-6 type cytokine receptors.

The ObR mRNA transcript, which is about 5 kb long, is expressed in thechoroid plexus, the hypothalamus and other tissues, including lung andliver. The murine short forms described herein encode receptor proteinsof 894 (FIGS. 1A-1D) and 893 amino acids; murine long form obR cDNAs andhuman obR cDNAs, described herein, encode receptor proteins of 1162amino acids and 1165 amino acids, respectively (FIGS. 1A-1D and FIGS.3A-3F). The ObR has a typical hydrophobic leader sequence (about 22amino acids long in both forms of murine ObR, and about 20 amino acidslong in human ObR); an extracellular domain (about 815 amino acids longin both forms of murine ObR, and about 819 amino acids long in humanObR); a short transmembrane region (about 23 amino acids long in bothforms of murine ObR and human ObR); and a cytoplasmic domain. Thetranscripts encoding the murine ObR short (FIGS. 1A-1D) and long form(FIGS. 6A-6F) are identical until the fifth codon 5′ of the stop codonof the short form and then diverge completely, suggestive of alternativesplicing. As described herein, the cytoplasmic domain encoded by the 894amino acid murine short form obR cDNA is 34 amino acids, while thatencoded by the murine long form obR cDNA (302 amino acids) isapproximately the same length as the cytoplasmic domain encoded by thehuman obR cDNA (303 amino acids). The deduced amino acid sequences frommurine long form ObR and human ObR are homologous throughout the lengthof the coding region and share 75% identity (FIGS. 7A-7B).

The obese phenotype of the db mouse results from a G→T transversion inthe obR gene. This transversion creates a splice donor site which inturn leads to aberrant processing of obR long form mRNA in db mutants.In db mutants this aberrant processing generates long form mRNAs whichencode a truncated ObR protein that is identical to the 894 amino acidshort form ObR. Like the short form ObR, the mutant long form ObR lacksmost of the cytoplasmic domain and is incapable of transducing a signalvia a STAT mediated pathway. The signalling competant long form ObR,which is absent in the db/db mice, is required for body weightmaintenance.

The invention encompasses the following nucleotides, host cellsexpressing such nucleotides, and the expression products of suchnucleotides: (a) nucleotides that encode mammalian ObRs, including thehuman ObR, and the obR gene product; (b) nucleotides that encodeportions of the ObR that correspond to its functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the extracellular domain (ECD), the transmembranedomain (TM), and the cytoplasmic domain (CD); (c) nucleotides thatencode mutants of the ObR in which all or a part of one of the domainsis deleted or altered, and the polypeptide products specified by suchnucleotide sequences, including but not limited to soluble receptors inwhich all or a portion of the TM is deleted, and nonfunctional receptorsin which all or a portion of the CD is deleted; (d) nucleotides thatencode fusion proteins containing the ObR or one of its domains (e.g.,the extracellular domain) fused to another polypeptide.

The invention also encompasses agonists and antagonists of ObR,including small molecules, large molecules, mutant Ob proteins thatcompete with native Ob, and antibodies, as well as nucleotide sequencesthat can be used to inhibit obR gene expression (e.g., antisense andribozyme molecules, and gene or regulatory sequence replacementconstructs) or to enhance obR gene expression (e.g., expressionconstructs that place the obR gene under the control of a strongpromoter system), and transgenic animals that express an obR transgeneor “knock-outs” that do not express ObR.

In addition, the present invention encompasses methods and compositionsfor the diagnostic evaluation, typing and prognosis of body weightdisorders, including obesity and cachexia, and for the identification ofsubjects having a predisposition to such conditions. For example, obRnucleic acid molecules of the invention can be used as diagnostichybridization probes or as primers for diagnostic PCR analysis for theidentification of obR gene mutations, allelic variations and regulatorydefects in the obR gene. The present invention further provides fordiagnostic kits for the practice of such methods.

Further, the present invention also relates to methods for the use ofthe obR gene and/or obR gene products for the identification ofcompounds which modulate, i.e., act as agonists or antagonists, of obRgene expression and or obR gene product activity. Such compounds can beused as agents to control body weight and, in particular, as therapeuticagents for the treatment of body weight and body weight disorders,including obesity, cachexia and anorexia.

Still further, the invention encompasses methods and compositions forthe treatment of body weight disorders, including obesity, cachexia, andanorexia. Such methods and compositions are capable of modulating thelevel of obR gene expression and/or the level of obR gene productactivity.

This invention is based, in part, on the surprising discovery, after anextensive survey of numerous cell lines and tissues, of a high affinityreceptor for Ob in the choroid plexus of the brain, the identificationand cloning of obR cDNA from a library prepared from choroid plexusmRNA, characterization of its novel sequence, mapping the obR gene tothe same genetic interval in the mouse genome as the db gene maps, andcharacterization of the ObR as a transmembrane receptor of the Class Icytokine receptor family. obR mRNA was detected in other tissues,including the hypothalamus.

The full-length ObR, expressed predominantly in the hypothalamus signalstransduces through activation of STAT proteins and stimulation oftranscription through IL-6 responsive gene elements. The ability of thefull-length long form ObR to signal is in contrast to the naturallyoccurring truncated form or the mutant form found in db/db mice whichare unable to mediate signal transduction. The invention also includesforms of ObR lacking one or another of the intracellular domainsimportant for signalling and induction of gene expression.

3.1. DEFINITIONS

As used herein, the following terms, whether used in the singular orplural, will have the meanings indicated:

Ob: means the Ob protein described in Zhang, Y. et al., 1994, Nature372:425-432, which is incorporated herein by reference in its entirety,which is also known as leptin. Ob includes molecules that are homologousto Ob or which bind to ObR. Ob fusion proteins having an N-terminalalkaline phosphatase domain are referred to herein as AP-Ob fusionproteins, while Ob fusion proteins having a C-terminal alkalinephosphatase domain are referred to herein as Ob-AP fusion proteins.

obR nucleotides or coding sequences: means nucleotide sequences encodingObR protein, polypeptide or peptide fragments of ObR protein, or ObRfusion proteins. obR nucleotide sequences encompass DNA, includinggenomic DNA (e.g. the obR gene) or cDNA, or RNA.

ObR: means Ob receptor protein. Polypeptides or peptide fragments of ObRprotein are referred to as ObR polypeptides or ObR peptides. Fusions ofObR, or ObR polypeptides or peptide fragments to an unrelated proteinare referred to herein as ObR fusion proteins.

A functional ObR refers to a protein which binds Ob with high affinityin vivo or in vitro.

ECD: means “extracellular domain”.

TM: means “transmembrane domain”.

CD: means “cytoplasmic domain”.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of murine obR (short form) cDNA encoding murineshort form ObR protein (894 amino acids). The domains of short formmurine ObR are: signal sequence (amino acid residue 1 to about aminoacid residue 22), extracellular domain (from about amino acid residue 23to about amino acid residue 837), transmembrane domain (from about aminoacid residue 838 to about amino acid residue 860), and cytoplasmicdomain (from about amino acid residue 861 to about amino acid residue894). Potential N-linked glycosylation sites in the extracellular domainare indicated by asterisks above the first amino acid of the N-X-S andN-X-T motifs. Underscore indicate motifs conserved in the class Icytokine receptor family.

FIG. 2. AP-Ob fusion protein binding studies.

FIG. 2A. COS-7 cells transfected with the ObR cDNA were treated withvarious AP or AP-Ob fusion proteins at 1 nM (diluted in DMEM+10% FBS).Columns show the average of two binding determinations and error barsshow the difference between the two. 1) Unfused AP, 2) AP-Ob (mouse), 3)AP-Ob (mouse)+100 nM mouse Ob, 4) AP-Ob (mouse)+100 nM human Ob, 5)AP-Ob (human), 6) Ob-AP (mouse), 7) AP-Ob (mouse) incubated with mocktransfected (vector- no insert) COS-7 cells.

FIG. 2B-1. Binding isotherm and Scatchard analysis of the interaction ofAP-Ob and ObR. COS-7 cells transfected with the obR cDNA were incubatedwith various concentrations of the AP-Ob (mouse) fusion protein FIG.2B-2. Scatchard transformation is shown as an inset.

FIGS. 3A-3F. Nucleotide sequence (SEQ ID NO:3) and deduced amino acidsequence (SEQ ID NO:4) of human obR cDNA encoding human ObR protein. Thedomains of human ObR are: signal sequence (from amino acid residue 1 toabout amino acid residue 20), extracellular domain (from about aminoacid residue 21 to about amino acid residue 839), transmembrane domain(from about amino acid residue 840 to about amino acid residue 862), andcytoplasmic domain (from about amino acid residue 863 to about aminoacid residue 1165). Also depicted are 5′ untranslated nucleotidesequences. Potential N-linked glycosylation sites in the extracellulardomain are indicated by asterisks above the first amino acid of theN-X-S and N-X-T motifs. Underscores indicate motifs conserved in theclass I cytokine receptor family.

FIG. 4. Alignment of the extracellular domains of the murine ObR andhuman gp130. Identical residues (black) and conservative changes (gray)are indicated by shading around the corresponding amino acids.Conservative changes indicated are as defined by FASTA.

FIGS. 5A-5B. Alignment of mouse ObR (short form shown in FIGS. 1A-1D)and human ObR. Amino acids that are identical between the two sequencesare indicated by a star

FIGS. 6A-6F. Nucleotide sequence and deduced amino acid sequence (SEQ IDNO:43) of murine long form obR cDNA encoding murine long form ObRprotein. The domains of long form murine ObR are: signal sequence (aminoacid residue 1 to about amino acid residue 22), extracellular domain(from about amino acid residue 23 to about amino acid residue 837),transmembrane domain (from about amino acid residue 838 to about aminoacid residue 860), and cytoplasmic domain (from about amino acid residue861 to about amino acid residue 1162).

FIGS. 7A-7B. Alignment of the long forms of human and murine ObR.Identical residues and conservative changes are indicated by twoasterisks or one asterisk, respectively. Conservative changes indicatedare as defined by FASTA. Abbreviations: mobr-1, murine ObR long form;and hobr, human homolog.

FIG. 8. Location of the gene encoding ObR on mouse chromosome 4.

FIG. 9. Nucleotide sequence of the 106 base pair insert in the long formtranscript of db/db. The precise position of the insertion in thededuced amino acid sequence near the insertion region are shown.

FIG. 10. Bar graph depicting ObR-Ig neutralization of OB protein. COScell were transiently transfected with the ObR cDNA and tested for theirability to bind 0.5 nM AP-OB. Column 1 shows the high levels of specificbinding observed in the absence of ObR-IgG fusion protein. Columns 2, 3and 4 show the near complete inhibition of binding observed with threedifferent column fractions of purified ObR IgG.

FIG. 11A. Schematic drawings of various C-terminal deletion mutants ofObR protein. The names and predicted length (aa) of the proteins areshown above each protein. The extracellular domains are shown asstriped, the transmembrane domains are shown as black, and thecytoplasmic domains are shown as white. The location of tyrosineresidues in the cytoplasmic domain are indicated by horizontal bars (Y986, Y1079, and Y1141 are conserved between human and murine ObR). Thelength of the cytoplasmic domains (aa) are shown below each protein.

FIG. 11B. A bar graph depicting the results of CAT assays employing aIL-6RE-CAT expression construct (upper panel) or a HRRE-CAT expressionconstruct (lower panel) and the ObR deletion mutants of FIG. 11A. H-35cells were transfected with cDNAs encoding the ObR mutant and eitherIl-6RE-CAT or HRRE-CAT. Subcultures of cells were treated for 24 hourswith serum-free medium alone (−) or serum-free medium containing mouseleptin (+). CAT activity was determined and is expressed relative tovalues obtained for untreated control cultures.

FIG. 12. A bar graph depicting the results of an AP-Ob fusion proteinbinding assay. COS-7 cells were transfected with a cDNA encoding theindicated ObR protein. Forty-eight hours later cells were incubated with1 mM AP-Ob fusion protein. Bars show the average of two binding assays.The error bars indicate the difference between the two assays.

FIG. 13A. Schematic drawings of various mutant ObR proteins. Thelocation of tyrosine residues 986 and 1079 are indicated. The locationof the “box 1” sequence is also indicated.

FIG. 13B. Bar graph depicting the results of a HRRE-CAT induction assay.H-35 cells were co-transfected with HRRE-CAT and expression constructsfor either OB-RY986F, OB-RY1079F or OB-R(box 1 mt). Subcultures of cellswere treated for 24 h with serum-free medium containing human leptin.CAT activity was determined and is expressed relative to values obtainedfor untreated control cultures.

FIG. 14A. Schematic drawings of various receptor chimeras. The portionsderived from G-CSFR are shaded; the portions derived from ObR are not.The locations of the predicted Box 1, Box 2, and Box 3 motifs areindicated.

FIGS. 14B and 14C. Bar graphs depicting the results of IL-6RE-CAT (leftpanel) and HRRE-CAT induction assays. H-35 cells were co-transfectedwith expression plasmids for the indicated recpotr (ObR, G-CSFR, orchimeric) and IL-6-RE-CAT or HREE-CAT expression construct. Cells werestimulated with the appropriate ligand and CAT activity was determinedas in the experiments described in FIG. 11. All values are expressedrelative to untreated control cultures (mean±std deviation of 3 to 4experiments).

FIG. 15A. Bar graph depicting the results of HRRE-CAT induction assays.H-35 cells were co-transfected with HRRE-CAT and the indicated amount ofObR and OB-RΔ868-1165. Cells were stimulated with leptin, and CATactivity was determined as in the experiments described in FIG. 11. Allvalues are expressed relative to the untreated cultures.

FIG. 15B. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of ObR/G-CSFR and OB-RΔ868-1165. Cells were stimulated withleptin, and CAT activity was determined as in the experiments describedin FIG. 11. All values are expressed relative to the untreated cultures.

FIG. 15C. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of G-CSFR and G-CSFR(Δcyto). Cells were stimulated with G-CSF,and CAT activity was determined as in the experiments described in FIG.11. All values are expressed relative to the untreated. cultures.

FIG. 15D. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of G-CSFR/ObR and G-CSFR(Δcyto). Cells were stimulated withG-CSF, and CAT activity was determined as in the experiments describedin FIG. 11. All values are expressed relative to the untreated cultures.

FIG. 15E. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of ObR and OB-RY1141F. Cells were stimulated with leptin, and CATactivity was determined as in the experiments described in FIG. 11. Allvalues are expressed relative to the untreated cultures.

5. DETAILED DESCRIPTION OF THE INVENTION

ObR, described for the first time herein, is a novel receptor proteinthat participates in body weight regulation. ObR is a transmembraneprotein that spans the membrane once and belongs to the Class I familyof cytokine receptors, and is most closely related to the gp130 signaltransducing component of the IL-6 receptor, the G-CSF receptor, and theLIF receptor. Signal transduction is triggered by the binding of Ob tothe receptor. Neutralization of Ob, removal of Ob, or interference withits binding to ObR results in weight gain. ObR mRNA is detected in thechoroid plexus, and other tissues, including the hypothalamus.

The invention encompasses the use of obR nucleotides, ObR proteins andpeptides, as well as antibodies to the ObR (which can, for example, actas ObR agonists or antagonists), antagonists that inhibit receptoractivity or expression, or agonists that activate receptor activity orincrease its expression in the diagnosis and treatment of body weightdisorders, including, but not limited to obesity, cachexia and anorexiain animals, including humans. The diagnosis of an ObR abnormality in apatient, or an abnormality in the ObR signal transduction pathway, willassist in devising a proper treatment or therapeutic regimen. Inaddition, obR nucleotides and ObR proteins are useful for theidentification of compounds effective in the treatment of body weightdisorders regulated by the ObR.

In particular, the invention described in the subsections belowencompasses ObR, polypeptides or peptides corresponding to functionaldomains of the ObR (e.g., ECD, TM or CD), mutated, truncated or deletedObRs (e.g. an ObR with one or more functional domains or portionsthereof deleted, such as ΔTM and/or ΔCD), ObR fusion proteins (e.g. anObR or a functional domain of ObR, such as the ECD, fused to anunrelated protein or peptide such as an immunoglobulin constant region,i.e., IgFc), nucleotide sequences encoding such products, and host cellexpression systems that can produce such ObR products.

The invention also features Ob receptors having an amino acid sequencethat is substantially identical to a defined amino acid sequence.

By “substantially identical” is meant a polypeptide or nucleic acidhaving a sequence that is at least 85%, preferably 90%, and morepreferably 95% or more identical to the sequence of the reference aminoacid or nucleic acid sequence. For polypeptides, the length of thereference polypeptide sequence will generally be at least 16 aminoacids, preferably at least 20 amino acids, more preferably at least 25amino acids, and most preferably 35 amino acids. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705).

In the case of polypeptide sequences which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

The invention also encompasses antibodies and anti-idiotypic antibodies(including Fab fragments), antagonists and agonists of the ObR, as wellas compounds or nucleotide constructs that inhibit expression of the obRgene (transcription factor inhibitors, antisense and ribozyme molecules,or gene or regulatory sequence replacement constructs), or promoteexpression of ObR (e.g., expression constructs in which obR codingsequences are operatively associated with expression control elementssuch as promoters, promoter/enhancers, etc.). The invention also relatesto host cells and animals genetically engineered to express the humanObR (or mutants thereof) or to inhibit or “knock-out” expression of theanimal's endogenous ObR.

The ObR proteins or peptides, ObR fusion proteins, obR nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant ObRs or inappropriately expressed ObRs for thediagnosis of body weight disorders such as obesity, anorexia orcachexia. The ObR proteins or peptides, ObR fusion proteins, obRnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs effective in the treatment of such bodyweight disorders. The use of engineered host cells and/or animals mayoffer an advantage in that such systems allow not only for theidentification of compounds that bind to the ECD of the ObR, but canalso identify compounds that affect the signal transduced by theactivated ObR.

Finally, the ObR protein products (especially soluble derivatives suchas peptides corresponding to the ObR ECD, or truncated polypeptideslacking the TM domain) and fusion protein products (especially ObR-Igfusion proteins, i.e., fusions of the ObR or a domain of the ObR, e.g.,ECD, ΔTM to an IgFc), antibodies and anti-idiotypic antibodies(including Fab fragments), antagonists or agonists (including compoundsthat modulate signal transduction which may act on downstream targets inthe ObR signal transduction pathway) can be used for therapy of suchdiseases. For example, the administration of an effective amount ofsoluble ObR ECD, ΔTM ObR or an ECD-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the ObR ECD would “mopup” or “neutralize” endogenous Ob, and prevent or reduce binding andreceptor activation, leading to weight gain. Nucleotide constructsencoding such ObR products can be used to genetically engineer hostcells to express such ObR products in vivo; these genetically engineeredcells function as “bioreactors” in the body delivering a continuoussupply of the ObR, ObR peptide, soluble ECD or ΔTM or ObR fusion proteinthat will “mop up” or neutralize Ob. Nucleotide constructs encodingfunctional ObRs, mutant ObRs, as well as antisense and ribozymemolecules can be used in “gene therapy” approaches for the modulation ofObR expression and/or activity in the treatment of body weightdisorders. Thus, the invention also encompasses pharmaceuticalformulations and methods for treating body weight disorders.

The invention is based, in part, on the surprising discovery of a highaffinity receptor for Ob expressed at significant concentration in thechoroid plexus. This discovery was made possible by using a novelalkaline phosphatase/Ob (AP-Ob) fusion protein for in situ staining ofcells and tissue. Competition studies with unlabeled Ob confirmed thatthe in situ binding observed was specific for Ob. Murine obR cDNA wasidentified using AP-Ob fusion protein to screen an expression library ofcDNAs synthesized from murine choroid plexus mRNA and transientlytransfected into mammalian COS cells. A clone, famj5312, expressing theshort form of a high affinity receptor for Ob was identified andsequenced. Sequence analysis revealed that the obR cDNA and predictedamino acid sequence are novel sequences containing amino acid regionsindicating that ObR is a member of the Class I family of receptorproteins. Mapping studies described herein demonstrate that the obR genemaps to the db locus. The data presented herein demonstrate further thatthe db gene is a mutant obR gene, which expresses an aberrantly splicedobR long form message that encodes a protein identical to the short formmurine ObR. The famj5312 sequence was utilized to screen a human fetalbrain cDNA library, which resulted in the identification of a human obRcDNA clone fahj5312d, described herein. Oligonucleotide primers designedon the basis of the human cDNA sequence were used to clone the humangenomic DNA clone, h-obR-p87, also described herein. mRNA encoding themurine long form of ObR was cloned from murine hypothalamus usingdegenerate primers designed on the human ObR cytoplasmic domain.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1. THE ObR GENE

The cDNA sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ IDNO:2) of murine short form (894 amino acids long) and murine long formObR are shown in FIGS. 1A-1D and 6A-6F, respectively. The signalsequence of both murine short and long form ObR extends from amino acidresidue 1 to about amino acid residue 22 of FIGS. 1A-1D and 6A-6F,respectively; the extracellular domain of both forms of murine ObRextends from about amino acid residue 23 to about amino acid residue 837of FIGS. 1A-1D and 6A-6F; the transmembrane domain of both forms ofmurine ObR extends from about amino acid residue 838 to about amino acidresidue 860 of FIGS. 1A-1D and 6A-6F; and the cytoplasmic domain of themurine short form ObR extends from about amino acid residue 861 to aboutamino acid residue 894 of FIGS. 1A-1D, while that of the long formextends from amino acid residue 861 to about amino acid residue 1162 ofFIGS. 6A-6F. At least one other short form of murine ObR has beenidentified, which is one amino acid shorter (i.e., 893 amino acids) thanthe sequence shown in FIGS. 1A-1D. The sequence at the C-terminusdiffers from the sequence shown in FIGS. 1A-1D, in that residues 890-894(RTDTL) are not present; and instead, residues 890-893 of the secondshort form have the following sequence: IMWI.

The cDNA sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ IDNO:4) of human ObR are shown in FIGS. 3A-3F. The human ObR signalsequence extends from amino acid residue 1 to about amino acid residue20 of FIGS. 3A-3F; the extracellular domain of human ObR extends fromabout amino acid residue 21 to about amino acid resiude 839 of FIGS.3A-3F; the transmembrane domain of human ObR extends from about aminoacid residue 840 to about amino acid residue 862 of FIGS. 3A-3F; and thecytoplasmic domain of human ObR extends from about amino acid residue863 to about amino acid residue 1165 of FIGS. 3A-3F. Sequences derivedfrom the human cDNA clone were used to design primers that were used toclone the human genomic obR, h-obR-p87, as described in the examples,infra.

Data presented in the working examples, infra, demonstrate that the obRgene maps to the db locus, and that the db gene is a mutant obR genewhich is expressed in db mice as an aberrantly spliced transcriptresulting in an mRNA species containing an insert of approximately 106nucleotides (nt) in the portion encoding the cytoplasmic domain of ObR.The insert produces a mutation that results in a transcript that encodesa prematurely truncated long form that is identical to murine short formObR.

The obR nucleotide sequences of the invention include: (a) the DNAsequence shown in FIGS. 1A-1D, 3A-3F or 6A-6F or contained in the cDNAclone famj5312 within E. coli strain 5312B4F3 as deposited with theAmerican Type Culture Collection (ATCC), or contained in the cDNA clonefahj5312d within E. coli strain h-obRD as deposited with the ATCC, orcontained in the human genomic clone, h-obR-p87 as deposited with theATCC; (b) nucleotide sequence that encodes the amino acid sequence shownin FIGS. 1A-1D, 3A-3F or 6A-6F, or the ObR amino acid sequence encodedby the cDNA clone famj5312 as deposited with the ATCC, or the cDNA clonefahj5312d as deposited with the ATCC, or contained in the human genomicclone, h-obR-p87 as deposited with the ATCC; (c) any nucleotide sequencethat hybridizes to the complement of the DNA sequence shown in FIGS.1A-1D, 3A-3F or 6A-6F or contained in the cDNA clone famj5312 asdeposited with the ATCC, or contained in the cDNA clone fahj5312d asdeposited with the ATCC, or contained in the human genomic clone,h-obR-p87 as deposited with the ATCC under highly stringent conditions,for example, hybridization to filter-bound DNA in 0.5 M NaHPO4, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68EC (Ausubel F. M. et al., eds., 1989, CurrentProtocols in Molecular Biology, Vol. 1, Green Publishing Associates,Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3) and encodes afunctionally equivalent gene product; and (d) any nucleotide sequencethat hybridizes to the complement of the DNA sequences that encode theamino acid sequence shown in FIGS. 1A-1D, 3A-3F or 6A-6F contained incDNA clone famj5312 as deposited with the ATCC, or contained in the cDNAclone fahj5312d as deposited with the ATCC, or contained in the humangenomic clone, h-obR-p87 as deposited with the ATCC under less stringentconditions, such as moderately stringent conditions, for example,washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yetwhich still encodes a functionally equivalent obR gene product.Functional equivalents of the ObR include naturally occurring ObRpresent in other species, and mutant ObRs whether naturally occurring orengineered. The invention also includes degenerate variants of sequences(a) through (d).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thenucleotide sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17- baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may encode or act as obR antisensemolecules, useful, for example, in obR gene regulation (for and/or asantisense primers in amplification reactions of obR gene nucleic acidsequences). With respect to obR gene regulation, such techniques can beused to regulate, for example, cachexia and/or anorexia. Further, suchsequences may be used as part of ribozyme and/or triple helix sequences,also useful for obR gene regulation. Still further, such molecules maybe used as components of diagnostic methods whereby, for example, thepresence of a particular obR allele responsible for causing a weightdisorder, such as obesity, may be detected.

In addition to the obR nucleotide sequences described above, full lengthobR cDNA or gene sequences present in the same species and/or homologsof the obR gene present in other species can be identified and readilyisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. The identification of homologs of obRin related species can be useful for developing animal model systemsmore closely related to humans for purposes of drug discovery. Forexample, expression libraries of cDNAs synthesized from choroid plexusmRNA derived from the organism of interest can be screened using labeledOb derived from that species, e.g., an AP-Ob fusion protein.Alternatively, such cDNA libraries, or genomic DNA libraries derivedfrom the organism of interest can be screened by hybridization using thenucleotides described herein as hybridization or amplification probes.Furthermore, genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of the obRgene product can also be identified via similar techniques. In the caseof cDNA libraries, such screening techniques can identify clones derivedfrom alternatively spliced transcripts in the same or different species.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of the obRnucleotide sequence, as shown in FIGS. 1A-1D, 3A-3, or 6A-6F. Thehybridization washing conditions used should be of a lower stringencywhen the cDNA library is derived from an organism different from thetype of organism from which the labeled sequence was derived. Withrespect to the cloning of a human obR homolog, using murine obR probes,for example, hybridization can, for example, be performed at 65ECovernight in Church's buffer (7% SDS, 250 mM NaHPO₄, 2 μM EDTA, 1% BSA).Washes can be done with 2×SSC, 0.1% SDS at 65° C. and then at 0.1×SSC,0.1% SDS at 65° C.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, NewYork; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience, New York.

Alternatively, the labeled obR nucleotide probe may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. The identification andcharacterization of human genomic clones is helpful for designingdiagnostic tests and clinical protocols for treating body weightdisorders in human patients. For example, sequences derived from regionsadjacent to the intron/exon boundaries of the human gene can be used todesign primers for use in amplification assays to detect mutationswithin the exons, introns, splice sites (e.g. splice acceptor and/ordonor sites), etc., that can be used in diagnostics.

Further, an obR gene homolog may be isolated from nucleic acid of theorganism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the obR gene product disclosed herein. The template forthe reaction may be cDNA obtained by reverse transcription of mRNAprepared from, for example, human or non-human cell lines or tissue,such as choroid plexus, known or suspected to express an obR geneallele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an obR gene. The PCRfragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the obR gene, such as, for example,choroid plexus or brain tissue). A reverse transcription reaction may beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” withguanines using a standard terminal transferase reaction, the hybrid maybe digested with RNAase H, and second strand synthesis may then beprimed with a poly-C primer. Thus, cDNA sequences upstream of theamplified fragment may easily be isolated. For a review of cloningstrategies which may be used, see e.g., Sambrook et al., 1989, supra.

The obR gene sequences may additionally be used to isolate mutant obRgene alleles. Such mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes to thesymptoms of body weight disorders such as obesity, cachexia or anorexia.Mutant alleles and mutant allele products may then be utilized in thetherapeutic and diagnostic systems described below. Additionally, suchobR gene sequences can be used to detect obR gene regulatory (e.g.,promoter or promotor/enhancer) defects which can affect body weight.

A cDNA of a mutant obR gene may be isolated, for example, by using PCR,a technique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant obRallele, and by extending the new strand with reverse transcriptase. Thesecond strand of the cDNA is then synthesized using an oligonucleotidethat hybridizes specifically to the 5′ end of the normal gene. Usingthese two primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant obR allele to that of the normal obR allele, themutation(s) responsible for the loss or alteration of function of themutant obR gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry the mutant obR allele,or a cDNA library can be constructed using RNA from a tissue known, orsuspected, to express the mutant obR allele. The normal obR gene or anysuitable fragment thereof may then be labeled and used as a probe toidentify the corresponding mutant obR allele in such libraries. Clonescontaining the mutant obR gene sequences may then be purified andsubjected to sequence analysis according to methods well known to thoseof skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant obR allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal obR gene product, as described, below, inSection 5.3. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborPress, Cold Spring Harbor.) Additionally, screening can be accomplishedby screening with labeled Ob fusion proteins, such as, for example,AP-Ob or Ob-AP fusion proteins. In cases where an obR mutation resultsin an expressed gene product with altered function (e.g., as a result ofa missense or a frameshift mutation), a polyclonal set of antibodies toObR are likely to cross-react with the mutant ObR gene product. Libraryclones detected via their reaction with such labeled antibodies can bepurified and subjected to sequence analysis according to methods wellknown to those of skill in the art.

The invention also encompasses nucleotide sequences that encode mutantObRs, peptide fragments of the ObR, truncated ObRs, and ObR fusionproteins. These include, but are not limited to nucleotide sequencesencoding mutant ObRs described in section 5.2 infra; polypeptides orpeptides corresponding to the ECD, TM and/or CD domains of the ObR orportions of these domains; truncated ObRs in which one or two of thedomains is deleted, e.g., a soluble ObR lacking the TM or both the TMand CD regions, or a truncated, nonfunctional ObR lacking all or aportion of the CD region. Nucleotides encoding fusion proteins mayinclude by are not limited to full length ObR, truncated ObR or peptidefragments of ObR fused to an unrelated protein or peptide, such as forexample, a transmembrane sequence, which anchors the ObR ECD to the cellmembrane; an Ig Fc domain which increases the stability and half life ofthe resulting fusion protein (e.g., ObR-Ig) in the bloodstream; or anenzyme, fluorescent protein, luminescent protein which can be used as amarker.

The invention also encompasses (a) DNA vectors that contain any of theforegoing ObR coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingObR coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing ObR codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors.

5.2. ObR PROTEINS AND POLYPEPTIDES

ObR protein, polypeptides and peptide fragments, mutated, truncated ordeleted forms of the ObR and/or ObR fusion proteins can be prepared fora variety of uses, including but not limited to the generation ofantibodies, as reagents in diagnostic assays, the identification ofother cellular gene products involved in the regulation of body weight,as reagents in assays for screening for compounds that can be used inthe treatment of body weight disorders, and as pharmaceutical reagentsuseful in the treatment of body weight disorders related to the ObR.

FIGS. 1A-1D and 6A-6F show the amino acid sequence of a murine shortform and long form ObR protein, respectively. In both of these forms ofObR, the signal sequence extends from amino acid 1 to about amino acid22; the ECD extends from about amino acid 23 to about amino acid 837;and the TM extends from about amino acid 838 to about amino acid 860. Inthe short form of murine ObR, the CD extends from about amino acid 861to about amino acid 894 (or to 893 in the second short form), while inthe long form it extends from about amino acid 861 to about amino acid1162. FIGS. 3A-3F show the amino acid sequence of a human ObR. Thesignal sequence extends from amino acid residue 1 to about amino acidresidue 20; the ECD extends from about amino acid residue 21 to aboutamino acid residue 839; the TM extends from about amino acid residue 840to about amino acid residue 862; and the CD extends from about aminoacid residue 863 to about amino acid residue 1165.

The ObR sequence begins with a methionine in a DNA sequence contextconsistent with a translation initiation site, followed by a typicalhydrophobic signal sequence of peptide secretion. The predicted matureextracellular domain for both forms of murine ObR is identical and is815 amino acids long, whereas the ECD predicted for human ObR is 819amino acids long. The extracellular domain of ObR shows many features ofthe class I cytokine receptor family (reviewed in Heldin, 1995, Cell80:213-223), and is most closely related to the gp13o signal transducingcomponent of the IL-6 receptor (Taga et al., 1989, Cell 58:573-581), theG-CSF receptor (Fukunaga et al., 1990, Cell 61:341-350), and the LIFreceptor (Gearing et al., 1991, Science 255:1434-1437). In fact, thedata presented herein demonstrate that the long form ObR signals throughactivation of STAT proteins—a hallmark of the signal transductionpathway mediated by the IL-6 type cytokine receptor family.

An alignment between the extracellular domains of the murine ObR andgp130 is shown in FIG. 4. Although the overall amino acid sequenceidentity between these two molecules is low (24%), thecharacteristically conserved cysteine residues, the Trp-Ser-X-Trp-Sermotif (SEQ ID NO:6; amino acid residues 317-321 and 620-624 in themurine sequence shown in FIGS. 1A-1D; amino acid residues 319-323 and622-626 in the human sequence shown in FIGS. 3A-3F), and conservation ofother residues within this group of proteins (reviewed in Kishimoto etal., 1994, Cell 76:253-262) is clearly evident. The amino acid sequencesof murine short form ObR and human ObR are highly homologous throughoutthe length of murine short form ObR (FIGS. 5A-5B). In fact, the deducedamino acid sequence identity between the murine short form and humanclones (78%) is the same or greater than that seen when comparing themurine and human forms of gp130 (Saito et al., 1992, J. Immunol.148:4066-4071), the LIF receptor (Gough et al., 1988, Proc. Natl. Acad.Sci. USA 85:2623-2627), and the G-CSF receptor (Fukanaga et al., 1990,Proc. Natl. Acad. Sci. USA 87:8702-8706). Similarly, the deduced aminoacid sequences from murine and human long forms of ObR are homologousthroughout the length of the coding region and share 75% identity (FIGS.7A-7B).

Potential N-linked glycosylation sites (i.e., amino acid sequence motifN-X-S or N-X-T) are found in the ECD of both murine and human ObR. Atleast twenty potential N-linked glycosylation sites can be identified inthe murine ObR ECD sequence shown in FIGS. 1A-1D and 6A-6F (seetripeptide motifs starting at amino acid residues 23, 41, 56, 73, 81,98, 187, 206, 276, 347, 397, 433, 516, 624, 659, 670, 688, 697, 728, and750); whereas at least sixteen potential N-linked glycosylation sitescan be identified in the human ObR ECD sequence shown in FIGS. 3A-3F(see tripeptide motifs starting at amino acid residues 41, 56, 73, 98,187, 275, 345, 431, 514, 622, 657, 668, 686, 695, 698 and 726). Theextracellular domain of both the murine and human ObR is followed by apredicted transmembrane domain of 23 amino acids.

The murine cDNA shown in FIGS. 1A-1D encodes a short cytoplasmic domain(34 amino acids). Amino acids 5-24 of the murine ObR cytoplasmic domain(i.e., amino acid residues 865 to 884 in FIGS. 1A-1D) show 47% identityto membrane proximal sequences of the intracellular domain of the LIFreceptor, and contain a box 1 Jak interaction sequence (Narazaki et al.,1994, Proc. Natl. Acad. Sci. USA 91:2285-2289). Interestingly, the humancDNA encodes a protein with a much longer intracellular domain thanmurine short form ObR. Although the murine short form and humanintracellular domains are highly conserved up to the final five residuesof murine short form ObR, the human intracellular domain continues to alength similar to that of gp130. The nucleotide sequences of the murineshort form and human clones are also very similar throughout the codingregion of murine short form ObR, but then diverge completely near themurine short form ObR stop codon.

The short cytoplasmic domain of the murine short form cDNAs describedherein is characteristic of several class I cytokine receptorpolypeptides (reviewed in Kishimoto et al., 1994, Cell 76:253-262).However, the results reported herein demonstrate that the short form ObRdoes not activate signal transduction via the STAT pathway which ismediated by the long form ObR. In fact, the three receptors to which ObRshows the strongest homology all have long cytoplasmic domains importantin intracellular signaling. This opened the possibility that the murineshort form ObR clone isolated was chimeric or encoded a rare aberrantlyspliced form not representing the major form expressed within thechoroid plexus. To address this issue, eight murine clones were selectedthat were independently identified in the library screen, and each wasamplified (in subpools of 150 clones each) by PCR with primers made tosequences 3′ of the stop codon. Results verified that all eight clonescontained these same 3′ untranslated sequences. In addition, theC-terminus of five independently isolated clones was sequenced and allshown to have the same stop codon. Finally, reverse transcription PCRwith total RNA from choroid plexus isolated from a mouse strain(C57Bl/KsJ) different from that which the cDNA library was derived,generated an identical PCR product containing a stop codon in the samelocation. These data indicated that the isolated murine short form cloneis neither chimeric nor a rare aberrant splice event, but rather islikely to be the predominant form of this receptor in the murine choroidplexus. The data presented herein indicate that in some tissues,alternatively spliced forms of mouse ObR exist with longer intracellulardomains (the long form); i.e., the wild-type obR gene is expressed intwo forms, one mRNA transcript having an insert of about 100 nucleotidesencodes ObR having a short cytoplasmic domain, and another mRNAtranscript encodes ObR having a long cytoplasmic domain that ishomologous to the human CD.

The murine cDNA shown in FIGS. 6A-6F encodes the long form ObR. Asdescribed supra, the amino acids encoding the ECD and TM of the murinelong form ObR are identical to those for the murine short form. Themurine long form cDNA, however, encodes a cytoplasmic domain (302 aminoacids) that is approximately the same length as the cytoplasmic domainencoded by the human ObR cDNA. Unlike the ObR short forms, the ObRencoded by the nucleotide sequence of the murine long form continues tobe similar to that of the human ObR throughout the cytoplasmic domain.

The data presented herein also indicate that db is a mutant of the longform murine obR gene. The db mutant expresses an aberrantly splicedtranscript containing an insert of about 106 nucleotides in the portionof the mRNA encoding the CD. Although the transcript is long, theinserted sequence produces a mutation that results in a transcript thatencodes a truncated ObR protein that is identical to the short forms ofObR and therefore, lacks most of the CD. The data shown hereindemonstrate that, unlike the long form ObR, the short form ObR, i.e.,the form of the receptor associated with the obese phenotype in db/dbmice, does not transduce signal mediated by the STAT pathway. Therefore,it appears that the signalling-competant long form ObR is activelyinvolved in body weight regulation and maintenance.

In sum, messenger RNA for several major ObR forms have been identified.The predominant ObR mRNA found in most tissues encodes a transmembraneprotein with a short cytoplasmic domain of 34 amino acid residuesreferred to as the short form. In hypothalamus, an obR mRNA exists whichencodes a protein with an identical extracellular domain as the shortform, but with a 302 residue-long cytoplasmic domain, referred to as thelong form. The db mutation leads to the production of an aberrant spliceproduct of long form transcript, resulting in a protein with truncatedcytoplasmic domain. Interestingly, the mRNA for the long form of ObR inthe db/db mice encodes a protein with an identical structure to thenaturally occurring short form. The loss of this carboxyterminal regionis proposed to render the ObR inactive and is predicted to generate theobese phenotype in db/db mice.

Sequence information indicated that ObR might exert a signaling actionsimilar to that of G-CSFR, LIFR and gp130 (Stahl & Yancopoulos, 1993,Cell 74:587-590; Kishimoto, et al., 1995, Blood 86:1243-1254). Signalingby these receptors entails, among others, the activation ofreceptor-associated kinases of the Janus kinase family which contributeto the phosphorylation and activation of the DNA binding activity ofSTAT1, STAT3 and STAT5 (Ihle, 1995, Nature 377:591-594; Kishimoto, etal., 1995, Blood 86:1243-1254). This process, in turn, has beencorrelated with induced transcription of genes that contain bindingsites for the STAT proteins such as the hepatic genes encoding acutephase plasma proteins (Lai et al., 1995, J. Biol. Chem.270:23254-23257). To address whether the cloned ObR isoforms are indeedsignaling receptor molecules, ObR was introduced into established tissueculture cell lines and the cell response to OB treatment was comparedwith that mediated by the structurally-related IL-6-type cytokinereceptors. The results presented in the example infra demonstrate thatthe long form ObR is a signal-transducing molecule. In particular, theresults show that the long form ObR shares functional specificity withIL-6-type cytokine receptors. The results also show that the short formObR does not signal via the STAT pathway transduced by the ObR longform. Thus, it appears that the long form ObR, but not the short form,is involved in maintenance of body weight.

The ObR amino acid sequences of the invention include the amino acidsequence shown in FIGS. 1A-1D (SEQ ID NO:2), FIGS. 3A-3F (SEQ ID NO:4)or FIGS. 6A-6F, or the amino acid sequence encoded by cDNA clonefamj5312 as deposited with the ATCC, or encoded by cDNA clone fahj5312das deposited with the ATCC, or encoded by the human genomic cloneh-obR-p87, as deposited with the ATCC. Further, ObRs of other speciesare encompassed by the invention. In fact, any ObR protein encoded bythe obR nucleotide sequences described in Section 5.1, above, are withinthe scope of the invention.

The invention also encompasses proteins that are functionally equivalentto the ObR encoded by the nucleotide sequences described in Section 5.1,as judged by any of a number of criteria, including but not limited tothe ability to bind Ob, the binding affinity for Ob, the resultingbiological effect of Ob binding, e.g., signal transduction, a change incellular metabolism (e.g., ion flux, tyrosine phosphorylation) or changein phenotype when the ObR equivalent is present in an appropriate celltype (such as the amelioration, prevention or delay of the obesephenotype, i.e., the db or ob phenotype), or weight loss. Suchfunctionally equivalent ObR proteins include but are not limited toadditions or substitutions of amino acid residues within the amino acidsequence encoded by the obR nucleotide sequences described, above, inSection 5.1, but which result in a silent change, thus producing afunctionally equivalent gene product. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid.

While random mutations can be made to obR DNA (using random mutagenesistechniques well known to those skilled in the art) and the resultingmutant ObRs tested for activity, site-directed mutations of the obRcoding sequence can be engineered (using site-directed mutagenesistechniques well known to those skilled in the art) to generate mutantObRs with increased function, e.g., higher binding affinity for Ob,and/or greater signalling capacity; or decreased function, e.g., lowerbinding affinity for Ob, and/or decreased signal transduction capacity.

For example, the alignment of mouse short form ObR FIGS. 1A-1D and thehuman ObR homolog FIGS. 3A-3F is shown in FIGS. 5A-5B in which identicalamino acid residues are indicated by a star. Mutant ObRs can beengineered so that regions of identity (indicated by stars in FIGS.5A-5B) are maintained, whereas the variable residues (unstarred in FIGS.5A-5B) are altered, for example, by deletion or insertion of an aminoacid residue(s) or by substitution of one or more different amino acidresidues. Conservative alterations at the variable positions can beengineered in order to produce a mutant ObR that retains function; forexample, Ob binding affinity or signal transduction capability or both.Non-conservative changes can be engineered at these variable positionsto alter function, for example, Ob binding affinity or signaltransduction capability, or both. Alternatively, where alteration offunction is desired, deletion or non-conservative alterations of theconserved regions (i.e., identical amino acids indicated by stars inFIGS. 5A-5B) can be engineered. For example, deletion ornon-conservative alterations (substitutions or insertions) of the CD,for example, amino acid residues 861-894 (FIGS. 1A-1D) of murine ObR, oramino acid residues 863-1165 (FIGS. 3A-3F) of human ObR, or portions ofthe CD, for example, amino acid residues 861-884 (FIGS. 1A-1D) of murineObR, or amino acid residues 863-886 (FIGS. 3A-3F) of human ObR (the box1 Jak interaction domain) can be engineered to produce a mutant ObR thatbinds Ob but is signalling-incompetent. Non-conservative alterations tothe starred residues in the ECD shown in FIGS. 5A-5B can be engineeredto produce mutant ObRs with altered binding affinity for Ob. The samemutation strategy can also be used to design mutant ObRs based on thealignment of murine long ObR form and the human ObR homolog shown inFIGS. 7A-7B in which identical amino acid residues are indicated by adouble asterisk.

FIG. 4 shows the alignment of the ECD of murine ObR with human gp130, inwhich identical residues are indicated in black, and conservativechanges are indicated in grey. Presumably, regions of identity andconservation are important for maintaining tertiary structure of theECD, whereas the variable regions may contribute to specificity of eachreceptor for its ligand. Therefore, ObR mutants with altered bindingaffinity for Ob may be engineered by altering the variable regions shownin FIG. 4. Such ObR mutants can be designed so as to preserve the ObRamino acid sequences that are boxed in FIG. 4 (both black and greyboxes) or to contain one or more conservative substitutions derived fromthe gp130 sequence shown in the grey boxes of FIG. 4.

Other mutations to the obR coding sequence can be made to generate ObRsthat are better suited for expression, scale up, etc. in the host cellschosen. For example, cysteine residues can be deleted or substitutedwith another amino acid in order to eliminate disulfide bridges;N-linked glycosylation sites can be altered or eliminated to achieve,for example, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionsof any one or more of the glycosylation recognition sequences whichoccur in the ECD (N-X-S or N-X-T), and/or an amino acid deletion at thesecond position of any one or more such recognition sequences in the ECDwill prevent glycosylation of the ObR at the modified tripeptidesequence. (See, e.g., Miyajima et al., 1986, EMBO J. 5(6):1193-1197).

Peptides corresponding to one or more domains of the ObR (e.g., ECD, TMor CD), truncated or deleted ObRs (e.g., ObR in which the TM and/or CDis deleted) as well as fusion proteins in which the full length ObR, anObR peptide or truncated ObR is fused to an unrelated protein are alsowithin the scope of the invention and can be designed on the basis ofthe obR nucleotide and ObR amino acid sequences disclosed in thisSection and in Section 5.1, above. Such fusion proteins include but arenot limited to IgFc fusions which stabilize the ObR protein or peptideand prolong half-life in vivo; or fusions to any amino acid sequencethat allows the fusion protein to be anchored to the cell membrane,allowing the ECD to be exhibited on the cell surface; or fusions to anenzyme, fluorescent protein, or luminescent protein which provide amarker function.

While the ObR polypeptides and peptides can be chemically synthesized(e.g., see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., New York), large polypeptides derivedfrom the ObR and the full length ObR itself may advantageously beproduced by recombinant DNA technology using techniques well known inthe art for expressing nucleic acid containing obR gene sequences and/orcoding sequences. Such methods can be used to construct expressionvectors containing the obR nucleotide sequences described in Section 5.1and appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encoding obRnucleotide sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to expressthe obR nucleotide sequences of the invention. Where the ObR peptide orpolypeptide is a soluble derivative (e.g., ObR peptides corresponding tothe ECD; truncated or deleted ObR in which the TM and/or CD are deleted)the peptide or polypeptide can be recovered from the culture, i.e., fromthe host cell in cases where the ObR peptide or polypeptide is notsecreted, and from the culture media in cases where the ObR peptide orpolypeptide is secreted by the cells. However, the expression systemsalso encompass engineered host cells that express the ObR or functionalequivalents in situ, i.e., anchored in the cell membrane. Purificationor enrichment of the ObR from such expression systems can beaccomplished using appropriate detergents and lipid micelles and methodswell known to those skilled in the art. However, such engineered hostcells themselves may be used in situations where it is important notonly to retain the structural and functional characteristics of the ObR,but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing obR nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the obR nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the obR sequences;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing obR nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the obR geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of ObR protein or for raising antibodies to the ObRprotein, for example, vectors which direct the expression of high levelsof fusion protein products that are readily purified may be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the obRcoding sequence may be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The obR gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of obR genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (E.g., see Smith et al., 1983, J. Virol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the obR nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the obR gene product in infected hosts. (E.g., See Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specificinitiation signals may also be required for efficient translation ofinserted obR nucleotide sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where an entire obRgene or cDNA, including its own initiation codon and adjacent sequences,is inserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the obR coding sequence is inserted, exogenoustranslational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (See Bittner et al., 1987, Methods inEnzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the-correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, choroid plexus cell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe obR sequences described above may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the obR geneproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the obR gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺· nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

The obR gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees may be used to generate obRtransgenic animals.

Any technique known in the art may be used to introduce the obRtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, −33T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the obRtransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the obR gene transgene beintegrated into the chromosomal site of the endogenous obR gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous obR gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous obR gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous obR gene in only that cell type,by following, for example, the teaching of Gu et al. (Gu, et al., 1994,Science 265: 103-106). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant obR gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of obR gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the obRtransgene product.

5.3. ANTIBODIES TO ObR PROTEINS

Antibodies that specifically recognize one or more epitopes of ObR, orepitopes of conserved variants of ObR, or peptide fragments of the ObRare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of the ObR in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of ObR. Such antibodies mayalso be utilized in conjunction with, for example, compound screeningschemes, as described, below, in Section 5.5, for the evaluation of theeffect of test compounds on expression and/or activity of the obR geneproduct. Additionally, such antibodies can be used in conjunction withthe gene therapy techniques described, below, in Section 5.6, to, forexample, evaluate the normal and/or engineered ObR-expressing cellsprior to their introduction into the patient. Such antibodies mayadditionally be used as a method for the inhibition of abnormal ObRactivity. Thus, such antibodies may, therefore, be utilized as part ofweight disorder treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the ObR, an ObR peptide (e.g., one corresponding the afunctional domain of the receptor, such as ECD, TM or CD), truncated ObRpolypeptides (ObR in which one or more domains, e.g., the TM or CD, hasbeen deleted), functional equivalents of the ObR or mutants of the ObR.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against obR gene products. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to the ObR can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” the ObR, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to the ObR ECD andcompetitively inhibit the binding of Ob to the ObR can be used togenerate anti-idiotypes that “mimic” the ECD and, therefore, bind andneutralize Ob. Such neutralizing anti-idiotypes or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens to neutralize Ob andpromote weight gain.

5.4. DIAGNOSIS OF BODY WEIGHT DISORDER ABNORMALITIES

A variety of methods can be employed for the diagnostic and prognosticevaluation of body weight disorders, including obesity, cachexia andanorexia, and for the identification of subjects having a predispositionto such disorders.

Such methods may, for example, utilize reagents such as the obRnucleotide sequences described in Section 5.1, and ObR antibodies, asdescribed, in Section 5.3. Specifically, such reagents may be used, forexample, for: (1) the detection of the presence of obR gene mutations,or the detection of either over- or under-expression of obR mRNArelative to the non-body weight disorder state; (2) the detection ofeither an over- or an under-abundance of obR gene product relative tothe non-body weight disorder state; and (3) the detection ofperturbations or abnormalities in the signal transduction pathwaymediated by ObR.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific obRnucleotide sequence or ObR antibody reagent described herein, which maybe conveniently used, e.g., in clinical settings, to diagnose patientsexhibiting body weight disorder abnormalities.

For the detection of obR mutations, any nucleated cell can be used as astarting source for genomic nucleic acid. For the detection of obR geneexpression or obR gene products, any cell type or tissue in which theobR gene is expressed, such as, for example, choroid plexus cells, maybe utilized.

Nucleic acid-based detection techniques are described, below, in Section5.4.1. Peptide detection techniques are described, below, in Section5.4.2.

5.4.1. DETECTION OF THE obR GENE AND TRANSCRIPTS

Mutations within the obR gene can be detected by utilizing a number oftechniques. Nucleic acid from any nucleated cell can be used as thestarting point for such assay techniques, and may be isolated accordingto standard nucleic acid preparation procedures which are well known tothose of skill in the art.

DNA may be used in hybridization or amplification assays of biologicalsamples to detect abnormalities involving obR gene structure, includingpoint mutations, insertions, deletions and chromosomal rearrangements.Such assays may include, but are not limited to, Southern analyses,single stranded conformational polymorphism analyses (SSCP), and PCRanalyses.

Such diagnostic methods for the detection of obR gene-specific mutationscan involve for example, contacting and incubating nucleic acidsincluding recombinant DNA molecules, cloned genes or degenerate variantsthereof, obtained from a sample, e.g., derived from a patient sample orother appropriate cellular source, with one or more labeled nucleic acidreagents including recombinant DNA molecules, cloned genes or degeneratevariants thereof, as described in Section 5.1, under conditionsfavorable for the specific annealing of these reagents to theircomplementary sequences within the obR gene. Preferably, the lengths ofthese nucleic acid reagents are at least 15 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid:obR molecule hybrid. The presence of nucleic acids which havehybridized, if any such molecules exist, is then detected. Using such adetection scheme, the nucleic acid from the cell type or tissue ofinterest can be immobilized, for example, to a solid support such as amembrane, or a plastic surface such as that on a microtiter plate orpolystyrene beads. In this case, after incubation, non-annealed, labelednucleic acid reagents of the type described in Section 5.1 are easilyremoved. Detection of the remaining, annealed, labeled obR nucleic acidreagents is accomplished using standard techniques well-known to thosein the art. The obR gene sequences to which the nucleic acid reagentshave annealed can be compared to the annealing pattern expected from anormal obR gene sequence in order to determine whether an obR genemutation is present.

Alternative diagnostic methods for the detection of obR gene specificnucleic acid molecules, in patient samples or other appropriate cellsources, may involve their amplification, e.g., by PCR (the experimentalembodiment set forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202),followed by the detection of the amplified molecules using techniqueswell known to those of skill in the art. The resulting amplifiedsequences can be compared to those which would be expected if thenucleic acid being amplified contained only normal copies of the obRgene in order to determine whether an obR gene mutation exists.

Additionally, well-known genotyping techniques can be performed toidentify individuals carrying obR gene mutations. Such techniquesinclude, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

Additionally, improved methods for analyzing DNA polymorphisms which canbe utilized for the identification of obR gene mutations have beendescribed which capitalize on the presence of variable numbers of short,tandemly repeated DNA sequences between the restriction enzyme sites.For example, Weber (U.S. Pat. No. 5,075,217, which is incorporatedherein by reference in its entirety) describes a DNA marker based onlength polymorphisms in blocks of (dC-dA)n-(dG-dT)n short tandemrepeats. The average separation of (dC-dA)n-(dG-dT)n blocks is estimatedto be 30,000-60,000 bp. Markers which are so closely spaced exhibit ahigh frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin the obR gene, and the diagnosis of diseases and disorders relatedto obR mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is incorporatedherein by reference in its entirety) describe a DNA profiling assay fordetecting short tri and tetra nucleotide repeat sequences. The processincludes extracting the DNA of interest, such as the obR gene,amplifying the extracted DNA, and labelling the repeat sequences to forma genotypic map of the individual's DNA.

The level of obR gene expression can also be assayed by detecting andmeasuring obR transcription. For example, RNA from a cell type or tissueknown, or suspected to express the obR gene, such as brain, especiallychoroid plexus cells, may be isolated and tested utilizing hybridizationor PCR techniques such as are described, above. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cells tobe used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of theobR gene. Such analyses may reveal both quantitative and qualitativeaspects of the expression pattern of the obR gene, including activationor inactivation of obR gene expression.

In one embodiment of such a detection scheme, cDNAs are synthesized fromthe RNAs of interest (e.g., by reverse transcription of the RNA moleculeinto cDNA). A sequence within the cDNA is then used as the template fora nucleic acid amplification reaction, such as a PCR amplificationreaction, or the like. The nucleic acid reagents used as synthesisinitiation reagents (e.g., primers) in the reverse transcription andnucleic acid amplification steps of this method are chosen from amongthe obR nucleic acid reagents described in Section 5.1. The preferredlengths of such nucleic acid reagents are at least 9-30 nucleotides. Fordetection of the amplified product, the nucleic acid amplification maybe performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product may be made suchthat the product may be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

Additionally, it is possible to perform such obR gene expression assays“in situ”, i.e., directly upon tissue sections (fixed and/or frozen) ofpatient tissue obtained from biopsies or resections, such that nonucleic acid purification is necessary. Nucleic acid reagents such asthose described in Section 5.1 may be used as probes and/or primers forsuch in situ procedures (See, for example, Nuovo, G. J., 1992, “PCR InSitu Hybridization: Protocols And Applications”, Raven Press, New York).

Alternatively, if a sufficient quantity of the appropriate cells can beobtained, standard Northern analysis can be performed to determine thelevel of mRNA expression of the obR gene.

5.4.2. DETECTION OF THE obR GENE PRODUCTS

Antibodies directed against wild type or mutant obR gene products orconserved variants or peptide fragments thereof, which are discussed,above, in Section 5.3, may also be used as body weight disorderdiagnostics and prognostics, as described herein. Such diagnosticmethods, may be used to detect abnormalities in the level of obR geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of the ObR, and may be performed invivo or in vitro, such as, for example, on biopsy tissue.

For example, antibodies directed to epitopes of the ObR ECD can be usedin vivo to detect the pattern and level of expression of the ObR in thebody. Such antibodies can be labeled, e.g., with a radio-opaque or otherappropriate compound and injected into a subject in order to visualizebinding to the ObR expressed in the body using methods such as X-rays,CAT-scans, or MRI. Labeled antibody fragments, e.g., the Fab or singlechain antibody comprising the smallest portion of the antigen bindingregion, are preferred for this purpose to promote crossing theblood-brain barrier and permit labeling ObRs expressed in the choroidplexus.

Additionally, any ObR fusion protein or ObR conjugated protein whosepresence can be detected, can be administered. For example, ObR fusionor conjugated proteins labeled with a radio-opaque or other appropriatecompound can be administered and visualized in vivo, as discussed, abovefor labeled antibodies. Further such Ob fusion proteins as AP-Ob onOb-Ap fusion proteins can be utilized for in vitro diagnosticprocedures.

Alternatively, immunoassays or fusion protein detection assays, asdescribed above, can be utilized on biopsy and autopsy samples in vitroto permit assessment of the expression pattern of the ObR. Such assaysare not confined to the use of antibodies that define the ObR ECD, butcan include the use of antibodies directed to epitopes of any of thedomains of the ObR, e.g., the ECD, the TM and/or CD. The use of each orall of these labeled antibodies will yield useful information regardingtranslation and intracellular transport of the ObR to the cell surface,and can identify defects in processing.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the obR gene, such as, forexample, choroid plexus cells. The protein isolation methods employedherein may, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cellsthat could be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of theobR gene.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.3, useful in the present invention may beused to quantitatively or qualitatively detect the presence of obR geneproducts or conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below, this Section) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Suchtechniques are especially preferred if such obR gene products areexpressed on the cell surface.

The antibodies (or fragments thereof) or Ob fusion or conjugatedproteins useful in the present invention may, additionally, be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immuno assays, for in situ detection of obR gene products orconserved variants or peptide fragments thereof, or for Ob binding (inthe case of labeled Ob fusion protein).

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody orfusion protein of the present invention. The antibody (or fragment) orfusion protein is preferably applied by overlaying the labeled antibody(or fragment) onto a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of the obRgene product, or conserved variants or peptide fragments, or Ob binding,but also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for obR gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofidentifying obR gene products or conserved variants or peptide fragmentsthereof, and detecting the bound antibody by any of a number oftechniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled ObR antibody or Obfusion protein. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody or fusion protein. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of ObR antibody or Ob fusion proteinmay be determined according to well known methods. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

With respect to antibodies, one of the ways in which the ObR antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.),1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect ObR through the use of aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then-determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

5.5. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE ObR EXPRESSION ORACTIVITY

The following assays are designed to identify compounds that interactwith (e.g., bind to) ObR (including, but not limited to the ECD or CD ofObR), compounds that interact with (e.g., bind to) intracellularproteins that interact with ObR (including, but not limited to, the TMand CD of ObR), compounds that interfere with the interaction of ObRwith transmembrane or intracellular proteins involved in ObR-mediatedsignal transduction, and to compounds which modulate the activity of obRgene (i.e., modulate the level of obR gene expression) or modulate thelevel of ObR. Assays may additionally be utilized which identifycompounds which bind to obR gene regulatory sequences (e.g., promotersequences) and which may modulate obR gene expression. See e.g., Platt,K. A., 1994, J. Biol. Chem. 269:28558-28562, which is incorporatedherein by reference in its entirety.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to peptides, antibodies and fragmentsthereof, and other organic compounds (e.g., peptidomimetics) that bindto the ECD of the ObR and either mimic the activity triggered by thenatural ligand (i.e., agonists) or inhibit the activity triggered by thenatural ligand (i.e., antagonists); as well as peptides, antibodies orfragments thereof, and other organic compounds that mimic the ECD of theObR (or a portion thereof) and bind to and “neutralize” natural ligand.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), andcombinatorial chemistry-derived molecular library made of D-and/or L-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able tocross the blood-brain barrier, gain entry into an appropriate cell(e.g., in the choroid plexus or in the hypothalamus) and affect theexpression of the obR gene or some other gene involved in the ObR signaltransduction pathway (e.g., by interacting with the regulatory region ortranscription factors involved in gene expression); or such compoundsthat affect the activity of the ObR (e.g., by inhibiting or enhancingthe enzymatic activity of the CD) or the activity of some otherintracellular factor involved in the ObR signal transduction pathway,such as, for example, gp130.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate ObR expression or activity. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be ligand binding sites, such as the interactiondomains of Ob with ObR itself. The active site can be identified usingmethods known in the art including, for example, from the amino acidsequences of peptides, from the nucleotide sequences of nucleic acids,or from study of complexes of the relevant compound or composition withits natural ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modelling can be used to completethe structure or improve its accuracy. Any recognized modelling methodmay be used, including parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a seach can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential ObR modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites ofOb, ObR, and related transduction and transcription factors will beapparent to those of skill in the art.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modelling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988, Acta PharmaceuticalFennica 97:159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinalyand Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perryand Davies, OSAR: Ouantitative Structure-Activity Relationships in DrugDesign pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc.R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a modelreceptor for nucleic acid components, Askew, et al., 1989, J. Am. Chem.Soc. 111:1082-1090. Other computer programs that screen and graphicallydepict chemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of the obRgene product, and for ameliorating body weight disorders. Assays fortesting the effectiveness of compounds, identified by, for example,techniques such as those described in Section 5.5.1 through 5.5.3, arediscussed, below, in Section 5.5.4.

5.5.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO ObR

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) ObR (including, but not limited to,the ECD or CD of ObR). Compounds identified may be useful, for example,in modulating the activity of wild type and/or mutant obR gene products;may be useful in elaborating the biological function of the ObR; may beutilized in screens for identifying compounds that disrupt normal ObRinteractions; or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to theObR involves preparing a reaction mixture of the ObR and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The ObR species usedcan vary depending upon the goal of the screening assay. For example,where agonists of the natural ligand are sought, the full length ObR, ora soluble truncated ObR, e.g., in which the TM and/or CD is deleted fromthe molecule, a peptide corresponding to the ECD or a fusion proteincontaining the ObR ECD fused to a protein or polypeptide that affordsadvantages in the assay system (e.g., labeling, isolation of theresulting complex, etc.) can be utilized. Where compounds that interactwith the cytoplasmic domain are sought to be identified, peptidescorresponding to the ObR CD and fusion proteins containing the ObR CDcan be used.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the ObRprotein, polypeptide, peptide or fusion protein or the test substanceonto a solid phase and detecting ObR/test compound complexes anchored onthe solid phase at the end of the reaction. In one embodiment of such amethod, the ObR reactant may be anchored onto a solid surface, and thetest compound, which is not anchored, may be labeled, either directly orindirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for ObR protein,polypeptide, peptide or fusion protein or the test compound to anchorany complexes formed in solution, and a labeled antibody specific forthe other component of the possible complex to detect anchoredcomplexes.

Alternatively, cell-based assays can be used to identify compounds thatinteract with ObR. To this end, cell lines that express ObR, or celllines (e.g., COS cells, CHO cells, fibroblasts, etc.) that have beengenetically engineered to express ObR (e.g., by transfection ortransduction of ObR DNA) can be used. Interaction of the test compoundwith, for example, the ECD of obR expressed by the host cell can bedetermined by comparison or competition with native Ob.

5.5.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE ObR

Any method suitable for detecting protein-protein interactions may beemployed for identifying transmembrane proteins or intracellularproteins that interact with ObR. Among the traditional methods which maybe employed are co-immunoprecipitation, crosslinking and co-purificationthrough gradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates and the ObR to identify proteins in thelysate that interact with the ObR. For these assays, the ObR componentused can be a full length ObR, a soluble derivative lacking themembrane-anchoring region (e.g., a truncated ObR in which the TM isdeleted resulting in a truncated molecule containing the ECD fused tothe CD), a peptide corresponding to the CD or a fusion proteincontaining the CD of ObR. Once isolated, such an intracellular proteincan be identified and can, in turn, be used, in conjunction withstandard techniques, to identify proteins with which it interacts. Forexample, at least a portion of the amino acid sequence of anintracellular protein which interacts with the ObR can be ascertainedusing techniques well known to those of skill in the art, such as viathe Edman degradation technique. (See, e.g., Creighton, 1983, “Proteins:Structures and Molecular Principles”, W. H. Freeman & Co., New York,pp.34-49). The amino acid sequence obtained may be used as a guide forthe generation of oligonucleotide mixtures that can be used to screenfor gene sequences encoding such intracellular proteins. Screening maybe accomplished, for example, by standard hybridization or PCRtechniques. Techniques for the generation of oligonucleotide mixturesand the screening are well-known. (See, e.g., Ausubel, supra., and PCRProtocols: A Guide to Methods and Applications, 1990, Innis, M. et al.,eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result in the simultaneousidentification of genes which encode the transmembrane or intracellularproteins interacting with ObR. These methods include, for example,probing expression, libraries, in a manner similar to the well knowntechnique of antibody probing of λgt11 libraries, using labeled ObRprotein, or an ObR polypeptide, peptide or fusion protein, e.g., an ObRpolypeptide or ObR domain fused to a marker (e.g., an enzyme, fluor,luminescent protein, or dye), or an Ig-Fc domain.

One method which detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to an obRnucleotide sequence encoding ObR, an ObR polypeptide, peptide or fusionprotein, and the other plasmid consists of nucleotides encoding thetranscription activator protein's activation domain fused to a cDNAencoding an unknown protein which has been recombined into this plasmidas part of a cDNA library. The DNA-binding domain fusion plasmid and thecDNA library are transformed into a strain of the yeast Saccharomycescerevisiae that contains a reporter gene (e.g., HBS or lacZ) whoseregulatory region contains the transcription activator's binding site.Either hybrid protein alone cannot activate transcription of thereporter gene: the DNA-binding domain hybrid cannot because it does notprovide activation function and the activation domain hybrid cannotbecause it cannot localize to the activator's binding sites. Interactionof the two hybrid proteins reconstitutes the functional activatorprotein and results in expression of the reporter gene, which isdetected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, ObR maybe used as the bait gene product. Total genomic or cDNA sequences arefused to the DNA encoding an activation domain. This library and aplasmid encoding a hybrid of a bait obR gene product fused to theDNA-binding domain are cotransfomed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene. For example, and not by way of limitation, a bait obRgene sequence, such as the open reading frame of obR (or a domain ofobR), as depicted in FIGS. 1A-1D, FIGS. 3A-3F, or FIGS. 6A-6F can becloned into a vector such that it is translationally fused to the DNAencoding the DNA-binding domain of the GAL4 protein. These colonies arepurified and the library plasmids responsible for reporter geneexpression are isolated. DNA sequencing is then used to identify theproteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait obR gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait obR gene-GAL4 fusion plasmid into a yeast strain which containsa lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait obR gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies which express HIS3 can be detected by their growthon petri dishes containing semi-solid agar based media lackinghistidine. The cDNA can then be purified from these strains, and used toproduce and isolate the bait obR gene-interacting protein usingtechniques routinely practiced in the art.

5.5.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH ObR/INTRACELLULAR ORObR/TRANSMEMBRANE MACROMOLECULE

INTERACTION

The macromolecules that interact with the ObR are referred to, forpurposes of this discussion, as “binding partners”. These bindingpartners are likely to be involved in the ObR signal transductionpathway, and therefore, in the role of ObR in body weight regulation.Therefore, it is desirable to identify compounds that interfere with ordisrupt the interaction of such binding partners with Ob which may beuseful in regulating the activity of the ObR and control body weightdisorders associated with ObR activity.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the ObR and its binding partneror partners involves preparing a reaction mixture containing ObRprotein, polypeptide, peptide or fusion protein as described in Sections5.5.1 and 5.5.2 above, and the binding partner under conditions and fora time sufficient to allow the two to interact and bind, thus forming acomplex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound may be initially included in the reactionmixture, or may be added at a time subsequent to the addition of the ObRmoiety and its binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the ObR moiety and the binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the ObR and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal ObR protein may also becompared to complex formation within reaction mixtures containing thetest compound and a mutant ObR. This comparison may be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal ObRs.

The assay for compounds that interfere with the interaction of the ObRand binding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the ObR moietyproduct or the binding partner onto a solid phase and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction bycompetition can be identified by conducting the reaction in the presenceof the test substance; i.e., by adding the test substance to thereaction mixture prior to or simultaneously with the ObR moiety andinteractive binding partner. Alternatively, test compounds that disruptpreformed complexes, e.g. compounds with higher binding constants thatdisplace one of the components from the complex, can be tested by addingthe test compound to the reaction mixture after complexes have beenformed. The various formats are described briefly below.

In a heterogeneous assay system, either the ObR moiety or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the obR gene product or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored may be used to anchor the species to the solid surface.The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the ObR moiety and theinteractive binding partner is prepared in which either the ObR or itsbinding partners is labeled, but the signal generated by the label isquenched due to formation of the complex (see, e.g., U.S. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation ofa signal above background. In this way, test substances which disruptObR/intracellular binding partner interaction can be identified.

In a particular embodiment, an ObR fusion can be prepared forimmobilization. For example, the ObR or a peptide fragment, e.g.,corresponding to the CD, can be fused to a glutathione-S-transferase(GST) gene using a fusion vector, such as pGEX-5X-1, in such a mannerthat its binding activity is maintained in the resulting fusion protein.The interactive binding partner can be purified and used to raise amonoclonal antibody, using methods routinely practiced in the art anddescribed above, in Section 5.3. This antibody can be labeled with theradioactive isotope ¹²⁵I, for example, by methods routinely practiced inthe art. In a heterogeneous assay, e.g., the GST-ObR fusion protein canbe anchored to glutathione-agarose beads. The interactive bindingpartner can then be added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between the obRgene product and the interactive binding partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-ObR fusion protein and the interactive bindingpartner can be mixed together in liquid in the absence of the solidglutathione-agarose beads. The test compound can be added either duringor after the species are allowed to interact. This mixture can then beadded to the glutathione-agarose beads and unbound material is washedaway. Again the extent of inhibition of the ObR/binding partnerinteraction can be detected by adding the labeled antibody and measuringthe radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the ObR and/or the interactive or binding partner (in cases where thebinding partner is a protein), in place of one or both of the fulllength proteins. Any number of methods routinely practiced in the artcan be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

For example, and not by way of limitation, an obR gene product can beanchored to a solid material as described, above, by making a GST-ObRfusion protein and allowing it to bind to glutathione agarose beads. Theinteractive binding partner can be labeled with a radioactive isotope,such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin.Cleavage products can then be added to the anchored GST-obR fusionprotein and allowed to bind. After washing away unbound peptides,labeled bound material, representing the intracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well-known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins usingrecombinant DNA technology.

5.5.4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE BODYWEIGHT DISORDERS

Compounds, including but not limited to binding compounds identified viaassay techniques such as those described, above, in Sections 5.5.1through 5.5.3, can be tested for the ability to ameliorate body weightdisorder symptoms, including obesity. The assays described above canidentify compounds which affect ObR activity (e.g., compounds that bindto the ObR, inhibit binding of the natural ligand, and either activatesignal transduction (agonists) or block activation (antagonists), andcompounds that bind to the natural ligand of the ObR and neutralizeligand activity); or compounds that affect obR gene activity (byaffecting obR gene expression, including molecules, e.g., proteins orsmall organic molecules, that affect or interfere with splicing eventsso that expression of the full length or the truncated form of the ObRcan be modulated). However, it should be noted that the assays describedcan also identify compounds that modulate ObR signal transduction (e.g.,compounds which affect downstream signalling events, such as inhibitorsor enhancers of tyrosine kinase or phosphatase activities whichparticipate in transducing the signal activated by Ob binding to theObR). The identification and use of such compounds which affect anotherstep in the ObR signal transduction pathway in which the obR gene and/orobR gene product is involved and, by affecting this same pathway maymodulate the effect of ObR on the development of body weight disordersare within the scope of the invention. Such compounds can be used aspart of a therapeutic method for the treatment of body weight disorders.

The invention encompasses cell-based and animal model-based assays forthe identification of compounds exhibiting such an ability to amelioratebody weight disorder symptoms. Such cell-based assay systems can also beused as the “gold standard” to assay for purity and potency of thenatural ligand, Ob, including recombinantly or synthetically produced Oband Ob mutants.

Cell-based systems can be used to identify compounds which may act toameliorate body weight disorder symptoms. Such cell systems can include,for example, recombinant or non-recombinant cells, such as cell lines,which express the obR gene. For example choroid plexus cells,hypothalamus cells, or cell lines derived from choroid plexus orhypothalamus can be used. In addition, expression host cells (e.g., COScells, CHO cells, fibroblasts) genetically engineered to express afunctional ObR and to respond to activation by the natural Ob ligand,e.g., as measured by a chemical or phenotypic change, induction ofanother host cell gene, change in ion flux (e.g., Ca⁺⁺), tyrosinephosphorylation of host cell proteins, etc., can be used as an end pointin the assay.

In utilizing such cell systems, cells may be exposed to a compoundsuspected of exhibiting an ability to ameliorate body weight disordersymptoms, at a sufficient concentration and for a time sufficient toelicit such an amelioration of body weight disorder symptoms in theexposed cells. After exposure, the cells can be assayed to measurealterations in the expression of the obR gene, e.g., by assaying celllysates for obR mRNA transcripts (e.g., by Northern analysis) or for obRprotein expressed in the cell; compounds which regulate or modulateexpression of the obR gene are good candidates as therapeutics.Alternatively, the cells are examined to determine whether one or morebody weight disorder-like cellular phenotypes has been altered toresemble a more normal or more wild type, non-body weight disorderphenotype, or a phenotype more likely to produce a lower incidence orseverity of disorder symptoms. Still further, the expression and/oractivity of components of the signal transduction pathway of which ObRis a part, or the activity of the ObR signal transduction pathway itselfcan be assayed.

For example, after exposure, the cell lysates can be assayed for thepresence of tyrosine phosphorylation of host cell proteins, as comparedto lysates derived from unexposed control cells. The ability of a testcompound to inhibit tyrosine phosphorylation of host cell proteins inthese assay systems indicates that the test compound inhibits signaltransduction initiated by ObR activation. The cell lysates can bereadily assayed using a Western blot format; i.e., the host cellproteins are resolved by gel electrophoresis, transferred and probedusing a anti-phosphotyrosine detection antibody (e.g., ananti-phosphotyrosine antibody labeled with a signal generating compound,such as radiolabel, fluor, enzyme, etc.) (See, e.g., Glenney et al.,1988, J. Immunol. Methods 109:277-285; Frackelton et al., 1983, Mol.Cell. Biol. 3:1343-1352). Alternatively, an ELISA format could be usedin which a particular host cell protein involved in the ObR signaltransduction pathway is immobilized using an anchoring antibody specificfor the target host cell protein, and the presence or absence ofphosphotyrosine on the immobilized host cell protein is detected using alabeled anti-phosphotyrosine antibody. (See, King et al., 1993, LifeSciences 53:1465-1472). In yet another approach, ion flux, such ascalcium ion flux, can be measured as an end point for ObR stimulatedsignal transduction.

Alternatively, activation of STAT proteins, and stimulation oftranscription mediated through IL-6 responsive gene elements may bemeasured to test the ability of a compound to regulate ObR mediatedsignal transduction. For example, a recombinant expression vector may beengineered to contain the IL6 responsive element sequences clonedadjacent to a reporter gene and regulation of ObR activity may bemeasured by assaying for reporter gene activity. Reporter genes that maybe used include, but are not limited to those encoding chloramphenicolacetyl transferase (CAT), firefly luciferase or human growth hormone.

In addition, animal-based body weight disorder systems, which mayinclude, for example, ob, db and ob/db mice, may be used to identifycompounds capable of ameliorating body weight disorder-like symptoms.Such animal models may be used as test substrates for the identificationof drugs, pharmaceuticals, therapies and interventions which may beeffective in treating such disorders. For example, animal models may beexposed to a compound, suspected of exhibiting an ability to amelioratebody weight disorder symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of body weight disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with body weight disorders such as obesity. With regard tointervention, any treatments which reverse any aspect of body weightdisorder-like symptoms should be considered as candidates for human bodyweight disorder therapeutic intervention. Dosages of test agents may bedetermined by deriving dose-response curves, as discussed in Section5.7.1, below.

5.6. THE TREATMENT OF BODY WEIGHT, INCLUDING BODY WEIGHT DISORDERS

The invention encompasses methods and compositions for modifying bodyweight and treating body weight disorders, including but not limited toobesity, cachexia and anorexia. Because a loss of normal obR geneproduct function results in the development of an obese phenotype, anincrease in obR gene product activity, or activation of the ObR pathway(e.g., downstream activation) would facilitate progress towards a normalbody weight state in obese individuals exhibiting a deficient level ofobR gene expression and/or obR activity.

Alternatively, symptoms of certain body weight disorders such as, forexample, cachexia, which involve a lower than normal body weightphenotype, may be ameliorated by decreasing the level of obR geneexpression, and/or obR gene activity, and/or downregulating activity ofthe ObR pathway (e.g., by targeting downstream signalling events).Different approaches are discussed below.

5.6.1. INHIBITION OF ObR EXPRESSION OR ObR ACTIVITY TO PROMOTE WEIGHTGAIN

Any method which neutralizes Ob or inhibits expression of the obR gene(either transcription or translation) can be used to effectuate weightgain. Such approaches can be used to treat body weight disorders such asanorexia or cachexia. Such methods can also be useful for agriculturalapplications; i.e., to increase the weight of livestock animals.

For example, the administration of soluble peptides, proteins, fusionproteins, or antibodies (including anti-idiotypic antibodies) that bindto and “neutralize” circulating Ob, the natural ligand for the ObR, canbe used to effectuate weight gain. To this end, peptides correspondingto the ECD of ObR, soluble deletion mutants of ObR (e.g., ΔTMObRmutants), or either of these ObR domains or mutants fused to anotherpolypeptide (e.g., an IgFc polypeptide) can be utilized. Alternatively,anti-idiotypic antibodies or Fab fragments of antiidiotypic antibodiesthat mimic the ObR ECD and neutralize Ob can be used (see Section 5.3,supra). Such ObR peptides, proteins, fusion proteins, anti-idiotypicantibodies or Fabs are administered to a subject in amounts sufficientto neutralize Ob and to effectuate weight gain.

ObR peptides corresponding to the ECD having the amino acid sequenceshown in FIGS. 1A-1D or 6A-6F, from about amino acid residue 23 to aboutamino acid residue 837, or having the amino acid sequence shown in FIGS.3A-3F, from about amino acid residue 21 to about amino acid residue 839,can be used. ObR ΔTM mutants in which all or part of the 23 amino acidhydrophobic anchor sequence (e.g., about amino acid residue 838 to aminoacid residue 860 in FIGS. 1A-1D or 6A-6F, or about amino acid residue840 to about amino acid residue 862 in FIGS. 3A-3F) could also be used.Fusion of the ObR, the ObR ECD or the ΔTMObR to an IgFc polypeptideshould not only increase the stability of the preparation, but willincrease the half-life and activity of the ObR-Ig fusion protein invivo. The Fc region of the Ig portion of the fusion protein can befurther modified to reduce immunoglobulin effector function. See Section10, infra.

In a specific embodiment described herein the extracellular domains ofthe mouse or human ObR were fused to the IgG constant region. Asindicated in FIG. 10, purified ObR-IgG was able to potently inhibit, orneutralize, the binding of the AP-OB fusion protein to cell surface ObR.(See Section 10.4.)

In an alternative embodiment for neutralizing circulating Ob, cells thatare genetically engineered to express such soluble or secreted forms ofObR may be administered to a patient, whereupon they will serve as“bioreactors” in vivo to provide a continuous supply of the Obneutralizing protein. Such cells may be obtained from the patient or anMHC compatible donor and can include, but are not limited tofibroblasts, blood cells (e.g., lymphocytes), adipocytes, muscle cells,endothelial cells etc. The cells are genetically engineered in vitrousing recombinant DNA techniques to introduce the coding sequence forthe ObR ECD, ΔTMObR, or for ObR-Ig fusion protein (e.g., ObR-, ECD- orΔTMObR-IgFc fusion proteins) into the cells, e.g, by transduction (usingviral vectors, and preferably vectors that integrate the transgene intothe cell genome) or transfection procedures, including but not limitedto the use of plasmids, cosmids, YACs, electroporation, liposomes, etc.The obR coding sequence can be placed under the control of a strongconstitutive or inducible promoter or promoter/enhancer to achieveexpression and secretion of the ObR peptide or fusion protein. Theengineered cells which express and secrete the desired ObR product canbe introduced into the patient systemically, e.g., in the circulation,intraperitoneally, at the choroid plexus or hypothalamus. Alternatively,the cells can be incorporated into a matrix and implanted in the body,e.g., genetically engineered fibroblasts can be implanted as part of askin graft; genetically engineered endothelial cells can be implanted aspart of a vascular graft. (See, for example, Anderson et al. U.S. Pat.No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each ofwhich is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous cells, they can beadministered using well known techniques which prevent the developmentof a host immune response against the introduced cells. For example, thecells may be introduced in an encapsulated form which, while allowingfor an exchange of components with the immediate extracellularenvironment, does not allow the introduced cells to be recognized by thehost immune system.

In an alternate embodiment, weight gain therapy can be designed toreduce the level of endogenous obR gene expression, e.g., usingantisense or ribozyme approaches to inhibit or prevent translation ofobR mRNA transcripts; triple helix approaches to inhibit transcriptionof the obR gene; or targeted homologous recombination to inactivate or“knock out” the obR gene or its endogenous promoter. Because the obRgene is expressed in the brain, including the choroid plexus andhypothalamus, delivery techniques should be preferably designed to crossthe blood-brain barrier (see PCT WO89/10134, which is incorporated byreference herein in its entirety). Alternatively, the antisense,ribozyme or DNA constructs described herein could be administereddirectly to the site containing the target cells; e.g., the choroidplexus and/or hypothalamus.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to ObR mRNA. The antisenseoligonucleotides will bind to the complementary obR mRNA transcripts andprevent translation. Absolute complementarity, although preferred, isnot required. A sequence “complementary” to a portion of an RNA, asreferred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double-stranded antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,for example, the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′- non- translated, non-coding regions of the obR shown in FIGS. 1A-1D(murine short form), FIGS. 6A-6F (murine long form) or FIGS. 3A-3F(human long form) could be used in an antisense approach to inhibittranslation of endogenous obR mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of ObR mRNA, antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects, the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides, or at least 50nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16: 3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85: 7448-7451), etc.

While antisense nucleotides complementary to the obR coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred. For example, antisenseoligonucleotides having the following sequences can be utilized inaccordance with the invention:

a) 5′-CATCTTACTTCAGAGAA-3′ (SEQ ID NO:7), which is complementary tonucleotides −14 to +3 in FIGS. 3A-3F;

b) 5′-CATCTTACTTCAGAGAAGTACAC-3′ (SEQ ID NO:8), which is complementaryto nucleotides −20 to +3 in FIGS. 3A-3F;

c) 5′-CATCTTACTTCAGAGAAGTACACCCATAA-3′ (SEQ ID NO:9), which iscomplementary to nucleotides −26 to +3 in FIGS. 3A-3F;

d) 5′-CATCTTACTTCAGAGAAGTACACCCATAATCCTCT-3′ (SEQ ID NO:10), which iscomplementary to nucleotides −32 to +3 in FIGS. 3A-3F;

e) 5′-AATCATCTTACTTCAGAGAAGTACACCCATAATCC-3′ (SEQ ID NO:11), which iscomplementary to nucleotides −29 to +6 in FIGS. 3A-3F;

f) 5′-CTTACTTCAGAGAAGTACACCCATAATCC-3′ (SEQ ID NO:12), which iscomplementary to nucleotides −29 to −1 in FIGS. 3A-3F;

g) 5′-TCAGAGAAGTACACCCATAATCC-3′ (SEQ ID NO:13), which is complementaryto nucleotides −29 to −7 in FIGS. 3A-3F;

h) 5′-AAGTACACCCATAATCC-3′ (SEQ ID NO:14), which is complementary tonucleotides −29 to −13 in FIGS. 3A-3F.

The antisense molecules should be delivered to cells which express theObR in vivo, e.g., the choroid plexus and/or hypothalamus. A number ofmethods have been developed for delivering antisense DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides. or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous obR transcripts and therebyprevent translation of the obR mRNA. For example, a vector can beintroduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systemically).

Ribozyme molecules designed to catalytically cleave obR mRNA transcriptscan also be used to prevent translation of obR mRNA and expression ofObR. (See, e.g., PCT International Publication WO90/11364, publishedOct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). Whileribozymes that cleave mRNA at site specific recognition sequences can beused to destroy obR mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature, 334:585-591. There are hundreds ofpotential hammerhead ribozyme cleavage sites within the nucleotidesequence of human obR cDNA (FIG. 3). Preferably the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the obR mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

For example, hammerhead ribozymes having the following sequences can beutilized in accordance with the invention:

a) 5′-ACAGAAUUUUUGACAAAUCAAAGCAGANNNNUCUGAGNAG UCCUUACUUCAGAGAA-3′ (SEQID NO:15), which will cleave human obR mRNA between nucleotides −1 and 1in FIGS. 3A-3F;

b) 5′-GGCCCGGGCAGCCUGCCCAAAGCCGGNNNNCCGGAGNAGUCG CCAGACCGGCUCGUG-3′ (SEQID NO:16), which will cleave between nucleotides −175 and −176 in FIGS.3A-3F;

c) 5′-UGGCAUGCAAGACAAAGCAGGNNNNCCUGAGNAGUCCUUAAAU CUCCAAGGAGUAA-3′ (SEQID NO:17), which will cleave between nucleotides 102 and 103 in FIGS.3A-3F;

d) 5′-UAUAUGACAAAGCUGUNNNNACAGAGNAGUCCUUGUGUGG UAAAGACACG3′ (SEQ IDNO:18), which will cleave between nucleotides 994 and 995 in FIGS.3A-3F;

e) 5′-AGCACCAAUUGAAUUGAUGGCCAAAGCGGGNNNNCCCGAGNAGU CAACCGUAACAGUAUGU-3′(SEQ ID NO:19), which will cleave between nucleotides 2142 and 2143 inFIGS. 3A-3F;

f) 5′-UGAAAUUGUUUCAGGCUCCAAAGCCGGNNNNCCGGAGNAGUCAAGAAGAGGACCACAUGUCACUGAUGC-3′ (SEQ ID NO:20), which will cleave betweennucleotides 2736 and 2737 in FIGS. 3A-3F;

g) 5′-GGUUUCUUCAGUGAAAUUACACAAAGCAGCNNNNGCUGAGNAGU CAGUUAGGUCACACAUC-3′(SEQ ID NO:21), which will cleave between nucleotides 3492 and 3493 inFIGS. 3A-3F;

h) 5′-ACCCAUUAUAACACAAAGCUGANNNNUCAGAGNAGUCAUCUG AAGGUUUCUUC-3′ (SEQ IDNO:22), which will cleave between nucleotides 3521 and 3522 in FIGS.3A-3F.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in obR.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the ObR in vivo, e.g.,hypothalamus and/or the choroid plexus. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous obR messages and inhibit translation. Because ribozymesunlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

Endogenous obR gene expression can also be reduced by inactivating or“knocking out” the obR gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional ObR (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenous obRgene (either the coding regions or regulatory regions of the obR gene)can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express ObR in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the obR gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive ObR (e.g., see Thomas & Capecchi 1987 and Thompson 1989,supra). However this approach can be adapted for use in humans providedthe recombinant DNA constructs are directly administered or targeted tothe required site in vivo using appropriate viral vectors, e.g., herpesvirus vectors for delivery to brain tissue; e.g., the hypothalamusand/or choroid plexus.

Alternatively, endogenous obR gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the obR gene (i.e., the obR promoter and/or enhancers) to formtriple helical structures that prevent transcription of the obR gene intarget cells in the body. (See generally, Helene, C. 1991, AnticancerDrug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y. Accad. Sci.,660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

In yet another embodiment of the invention, the activity of ObR can bereduced using a “dominant negative” approach to effectuate weight gain.To this end, constructs that encode defective ObRs can be used in genetherapy approaches to diminish the activity of the ObR in appropriatetarget cells. For example, nucleotide sequences that direct host cellexpression of ObRs in which the CD (e.g., FIGS. 1A-1D, amino acidresidues 861-894; FIGS. 6A-6F, amino acid residues 861-1162; or FIGS.3A-3F, amino acid residues 863-1165), or a portion of the CD (e.g., thebox I Jak interaction sequence; FIGS. 1A-1D and 6A-6F, amino acidresidues 861-884; or FIGS. 3A-3F, amino acid residues 863-886) isdeleted or mutated can be introduced into cells in the choroid plexus orhypothalamus (either by in vivo or ex vivo gene therapy methodsdescribed above). Alternatively, targeted homologous recombination canbe utilized to introduce such deletions or mutations into the subject'sendogenous obR gene in the hypothalamus or choroid plexus. Theengineered cells will express non-functional receptors (i.e., ananchored receptor that is capable of binding its natural ligand, butincapable of signal transduction). Such engineered cells present in thechoroid plexus or hypothalamus should demonstrate a diminished responseto the endogenous Ob ligand, resulting in weight gain.

5.6.2. RESTORATION OR INCREASE IN ObR EXPRESSION OR ACTIVITY TO PROMOTEWEIGHT LOSS

With respect to an increase in the level of normal obR gene expressionand/or ObR gene product activity, obR nucleic acid sequences can beutilized for the treatment of body weight disorders, including obesity.Where the cause of obesity is a defective ObR, treatment can beadministered, for example, in the form of gene replacement therapy.Specifically, one or more copies of a normal obR gene or a portion ofthe obR gene that directs the production of an obR gene productexhibiting normal function, may be inserted into the appropriate cellswithin a patient or animal subject, using vectors which include, but arenot limited to adenovirus, adeno-associated virus, retrovirus and herpesvirus vectors, in addition to other particles that introduce DNA intocells, such as liposomes.

Because the obR gene is expressed in the brain, including the choroidplexus and hypothalamus, such gene replacement therapy techniques shouldbe capable of delivering obR gene sequences to these cell types withinpatients. Thus, the techniques for delivery of the obR gene sequencesshould be designed to readily cross the blood-brain barrier, which arewell known to those of skill in the art (see, e.g., PCT application,publication No. WO89/10134, which is incorporated herein by reference inits entirety), or, alternatively, should involve direct administrationof such obR gene sequences to the site of the cells in which the obRgene sequences are to be expressed. Alternatively, targeted homologousrecombination can be utilized to correct the defective endogenous obRgene in the appropriate tissue; e.g., choroid plexus and/orhypothalamus. In animals, targeted homologous recombination can be usedto correct the defect in ES cells in order to generate offspring with acorrected trait.

Additional methods which may be utilized to increase the overall levelof obR gene expression and/or ObR activity include the introduction ofappropriate ObR-expressing cells, preferably autologous cells, into apatient at positions and in numbers which are sufficient to amelioratethe symptoms of body weight disorders, including obesity. Such cells maybe either recombinant or non-recombinant. Among the cells which can beadministered to increase the overall level of obR gene expression in apatient are normal cells, preferably choroid plexus cells, orhypothalamus cells which express the obR gene. The cells can beadministered at the anatomical site in the brain, or as part of a tissuegraft located at a different site in the body. Such cell-based genetherapy techniques are well known to those skilled in the art, see,e.g., Anderson, et al., U.S. Pat. No. 5,399,349; Mulligan & Wilson, U.S.Pat. No. 5,460,959.

Finally, compounds, identified in the assays described above, thatstimulate or enhance the signal transduced by activated ObR, e.g., byactivating downstream signalling proteins in the ObR cascade and therebyby-passing the defective ObR, can be used to achieve weight loss. Theformulation and mode of administration will depend upon thephysico-chemical properties of the compound. The administration shouldinclude known techniques that allow for a crossing of the blood-brainbarrier.

5.7. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The compounds that are. determined to affect obR gene expression or ObRactivity can be administered to a patient at therapeutically effectivedoses to treat or ameliorate weight disorders, including obesity,cachexia and anorexia. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptomsof body weight disorders.

5.7.1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.7.2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

6. EXAMPLE IN SITU LOCALIZATION OF ObR

In the Example presented herein, it is demonstrated via binding studieswith Ob (leptin)-alkaline phosphatase (AP) fusion proteins that highaffinity Ob receptor is present in mammalian choroid plexus tissue. Itis further demonstrated that the fusion protein binding observed wasOb-specific, and not due to a non-specific alkaline phosphatase-basedartifact.

6.1. MATERIALS AND METHODS

Construction and Expression of Ob-Alkaline Phosphatase Fusion Proteins.

Two types of fusion protein were generated. Specifically, Ob-AP fusionproteins were generated in which the AP portion was at the carboxylterminus of the fusion protein, and AP-Ob fusion proteins were generatedin which the AP portion was at the amino terminus of the fusion protein.

To produce mouse and human Ob-AP and AP-Ob fusion constructs, cDNAsequences were amplified by standard polymerase chain reactionprocedures. For mouse and human Ob-AP fusions, nucleotide sequencesencoding the entire open reading frames of mouse and human Ob,respectively were amplified from the corresponding cDNAs. Restrictionsites at the end of the amplification primers were cut with HindIII andBamHI (mouse) and inserted into the HindIII-BglII polylinker site ofAPtag-2, or BamHI and BglII (human) and inserted into the BglII site ofAPtag-2. For mouse and human AP-Ob fusion constructs, a new AP fusionvector expressing an AP molecule with its own signal peptide was firstgenerated (APtag-3) by replacing sequences between the HindIII and XhoIsites of APtag-2 with PCR amplified sequences of secreted placentalalkaline phosphatase (including signal sequence). A BglII site wasplaced so that fusions introduced into this site would be in-frame withthe AP protein. The sequences of the predicted mature forms of mouse andhuman Ob were then PCR amplified from the corresponding cDNAs.Restriction sites at the end of the amplification primers were cut withBamHI and BglII and inserted into the BglII site of APtag-3.

Each plasmid was transiently transfected into COS-7 cells (11.25 μg/150mm plate). Cells were grown to confluence and then media-conditioned for3 days. Cells were then centrifuged, 0.45 μm filtered, and stored at 4°C. with 20 mM Hepes (pH 7.0) and 0.05% sodium azide. Conditioned mediawere tested and quantitated for AP activity in a 96-well plate reader asdescribed by Flanagan and Leder (Flanagan, J. G. and Leder, P., 1990,Cell 63:185-194), except that homoarginine was omitted from all assays.

In Situ Fusion Protein Binding.

Quartered mouse brains, isolated choroid plexus, cells and cell lineswere rinsed once with HBHA (Hank's balanced salt solution with 0,5 mg/mlBSA, 0.1% NaN₃, 20 mM HEPES [pH 7.0]) in 12-well plates. Tissue was thenincubated with tissue culture supernatants containing AP-Ob fusion,Ob-AP fusion, or control supernatants (i.e., supernatants containingunfused AP only, containing AP-OB or OB-AP fusion proteins plus 80-foldmolar excess of E. coli-derived recombinant OB, or supernatants frommock-transfected COS cells), for 75 minutes with gentle rotation at roomtemperature. Samples were then treated as described previously (Cheng,H. J. and Flanagan, J. G., 1994, Cell 79:157-168).

6.2. RESULTS

To search for the Ob receptor, Ob-alkaline phosphatase fusion proteinswere constructed which would allow calorimetric detection of Ob binding.Specifically, cDNA molecules encoding the mouse and human Ob proteinswere inserted into the expression vectors APtag-2 and APtag-3, asdescribed, above in Section 6.1. Insertion into the expression vectorAPtag-2 resulted in a fusion protein with Ob at the N-terminus of thefusion protein and placental alkaline phosphatase (AP) at theC-terminus. The resulting fusion protein is referred to as Ob-AP.Insertion into the vector APtag-3 resulted in fusion proteins with AP atthe N-terminus fused to the predicted mature form of the Ob protein atthe C-terminus. The resulting fusion protein is referred to as AP-Ob.Both forms of murine fusion proteins were secreted and both wereproduced at the predicted molecular weight of approximately 81 kDa.

Several strategies were employed in an effort to identify cells ortissues expressing the Ob receptor. Each of the cells, cell lines andtissues tested as described herein were at least potentially involved inbody weight regulation. The first strategy employed was to attemptdirect binding assays with the Ob-AP and AP-Ob fusion proteins. Celllines examined by this strategy included the placental cell lines Be Wo(ATCC No. CCL98) and JAR (ATCC No. HTB144); the muscle cell lines L6(ATCC No. CRL1458) and BC3H (ATCC No. CRL1443); the neural cell linesPC12 (ATCC No. CRL1721) and NB41A3 (ATCC No. CCL147); the preadiposecell line 3T3-L1 (ATCC No. CRL173); and the liver cell line Hepa1-6(ATCC No. CRL1830). Also tested by this method were primary culturesfrom hypothalamus and primary cultures from cerebellum. None of thesestudies yielded positive binding results.

Second, attempts were made to identify cell lines expressing Ob receptorby examining changes in gene expression in response to the presence ofrecombinant Ob protein. The rationale here was that changes in geneexpression, whether obR gene expression or the expression of genesfurther downstream in the Ob/ObR-related signal transduction pathway,would identify cells in which ObR was present.

This analysis was done by standard differential display analysis (seePardee et al., U.S. Pat. No. 5,262,311) of RNA derived from Ob-treatedor untreated cells. Briefly, RNA was isolated from cells which eitherhad or had not been exposed to Ob, and was amplified via RT-PCR in amanner which allowed a direct quantitative comparison of the levels ofindividual transcripts present in the RNA derived from the Ob-treatedrelative to the Ob-untreated cell lines. Ob Cell lines tested by thisapproach were INS-1, 3T3-L1, Hepa1-6, L6, PC12, NB41A3 and BC3H. Inaddition, primary hypothalamic cultures were also tested. None of thecells tested exhibited a detectable quantitative difference inexpression pattern based on whether the cells had or had not beentreated with Ob.

Third, attempts to identify cells expressing Ob receptor were made bytreating cells with recombinant Ob protein and assaying for signs ofsignal transduction pathway activation. Specifically, cAMP changes weremonitored via ³H uptake, and tyrosine phosphorylation changes wereassayed via Western blots treated with anti-phosphotyrosine antibodies.Over twenty cell lines were examined in this manner. Specifically, thesecell lines included the mouse cell lines Y1 (adrenal cortex; ATCC No.CCL79), BC3H (smooth muscle-brain tumor; ATCC No. CRL1443), P19(embryonal carcinoma; ATCC No. CRL1825), 3T3L1 (preadipocyte; ATCC No.CRL173), Hepa1-6 (hepatoma; ATCC No. CRL1830), C2C12 (myoblast; ATCC No.CRL1772), NMUMG (mammary gland, normal epithelial; ATCC No. CRL1636),MM5MT (mammary gland; ATCC No. CRL1637), NB41A3 (neuroblastoma; ATCC No.CCL147), AtT20 (pituitary; ATCC No. CCL89), N MU LI (liver; ATCC No. CRL1638), BNL CL2 (liver; ATCC No. TIB73), and NCTC-1469 (liver; ATCC No.CCL91); rat cell lines, including L6 (myoblast; ATCC No. CRL1458), PC12(adrenal chromaffin; ATCC No. CRL1721), and H-4-II-E (hepatoma; ATCC No.CRL1548); and human cell lines, including SW872 (liposarcoma; ATCC No.HTB92), Hepa G2 (liver; ATCC No. HB8065), and neuroblastoma cell lines,including SK-N-SH (ATCC No. HTB11). Here again, no Ob-dependentdifferences were observed in any of the cells tested.

After an extensive search of mammalian cell lines and tissues, adultmouse brains were quartered, treated with AP-Ob fusion protein, washed,and tested for bound AP activity of the fusion protein usinghistological techniques, as described, above, in Section 6.1.Reproducible binding of the AP-Ob fusion protein was observed in therodent brain choroid plexus (within the lateral and third brainventricals). No AP-Ob staining was observed, however, in the braintissues surrounding the choroid plexus. The choroid plexus is a tissuelargely responsible for the generation of the cerebral spinal fluid.Further, choroid plexus tissue is considered to be one of the“guardians” of the blood-brain barrier.

Control AP staining was performed on tissues treated with unfused AP andon tissues which had been treated with AP-Ob in-the presence of anexcess of unfused Ob added to compete for the binding of the fusionprotein. Staining similar to that observed for the Ab-Ob fusion proteinwas not observed in either of these controls, demonstrating that theAP-Ob binding observed was Ob-specific, and not due to an AP-basedartifact.

In summary, therefore, only after employing several strategies, was acell surface molecule which binds Ob located; and this cell surfacemolecule was found within a specific region of the brain, the choroidplexus.

7. EXAMPLE: CLONING OF THE MURINE ObR GENE

Described, below, in Section 7.2.1, is the successful cloning of a shortform Ob receptor cDNA, famj5312, from expression libraries constructedusing murine choroid plexus RNA. The expression libraries were screenedusing AP-Ob fusion protein binding, as described, above, in the Examplepresented in Section 6. Section 7.2.2, below, describes the nucleotidesequence of the short form Ob receptor coding region and, further,describes the amino acid sequence of the Ob short form receptor protein.Section 7.2.3, below, describes competitive binding studiesdemonstrating that the protein encoded by the isolated cDNA encodes areceptor exhibiting high affinity binding for both mouse and human Obprotein. Section 7.2.4 describes studies which verify the authenticityof the isolated obR cDNA clone.

The high affinity Ob binding exhibited by the ObR, coupled with itshomology to the Class I family of cytokine receptors, as described,below, indicates that the ObR is involved in the control of mammalianbody weight, via signal transduction triggered by its binding to Obligand.

7.1. MATERIALS AND METHODS

Choroid Plexus mRNA Isolation.

Total RNA was isolated from 300 mouse choroid plexuses in batches of100, using the guanidinium isothiocyanate/CsCl method of Chirgwin et al.(1979, Biochemistry 18:5294) as described by R. Selden in CurrentProtocols for Molecular Biology (4.2.3 Supplement 14). Afterquantitation, the RNA was diluted to 1 mg/ml in distilled, deionizedwater and incubated for 30 min at 37° C. with an equal volume of DNasesolution (20 mM MgCl₂, 2 mM DTT, 0.1 units DNase, 0.6 units RNaseinhibitor in TE) to remove contaminating DNA. The RNA was extracted withphenol/chloroform/isoamyl, and ethanol precipitated. After quantitationat 260 nm, an aliquot was electrophoresed to check the integrity. Atotal of 320 μg of total RNA was purified.

Poly A+RNA was isolated using an Oligotex-dT kit (catalog #70042) fromQiagen (Chatsworth, Calif.) as described by the manufacturer. Afterquantitation, the mRNA was ethanol precipitated and resuspended at 1mg/ml in distilled, deionized, DEPC-treated water. A total of 11 μg ofpoly A+RNA was purified.

Library Construction. cDNA was synthesized according to the method ofGubler and Hoffman (Gene, 1983, 25:263) using a Superscript Plasmid cDNAsynthesis kit (Catalog #Series 8248) purchased from Life Technologies(Gaithersburg, Md.). The cDNA obtained was ligated into the NotI/Sal Isites of the mammalian expression vector pMET7, a modified version ofpME18S, which utilizes the SRα promoter as described previously (Takebe,Y. et al., 1988, Mol. Cel. Bio. 8:466). This vector was chosen becauseit contains a strong eukaryotic promoter, is expressed in COS7 cells,contains the AMP resistance gene, and is only 3.0 kb in length. Thesmall size of the vector is important because it increases theprobability of cloning large cDNAs. Other comparable vectors are 4.8 kband larger, thereby increasing the chances of imperfect replication, andreducing the probability of cloning large cDNAs. Ligated cDNA wasethanol precipitated and resuspended in distilled, deionized,DEPC-treated water at 25 ng/ml. One μl of the DNA was transformed byelectroporation per 40 μl of electrocompetent DH10B E. coli in a 0.1 cmcuvette.

cDNA was synthesized twice and used to construct two independent mousechoroid plexus libraries: mCP (mouse choroid plexus) A and mCP D.

DNA Preparation. Based on titers of the cDNA transformations,96-deepwell plates were inoculated with 150 cfu/well of primarytransformants in 1 ml of LB-amp. Primary transformants grown only 1 hourat 37° C. prior to aliquoting were used to avoid the overgrowth ofsmaller insert clones and thus underrepresentation of larger clones inthe 150 cfu pools. Cultures were grown 15-16 hours at 37° C. withaeration. Prior to prepping, 100 μl of cell suspension was removed andadded to 100 μl of 50% glycerol, mixed and stored at −80° C. (glycerolfreeze plate).

DNA was prepared using the Wizard™ Minipreps DNA Purification Systems(Promega, Madison, Wis.; Catalog No. A7100) employing modifications fora 96-well format. The protocol was as follows:

1) Cultures were centrifuged in 96-deepwell plate at 3200 rpm for 10minutes at 4° C. Supernatants were removed.

2) 140 μl each of cell resuspension solution (50 mM Tris-HCl, pH 7.5, 10mM EDTA, 100 μg/ml RNase A), cell lysis solution (0.2 M NaOH; 1.0% SDS)and neutralization solution (1.32 M Potassium acetate, pH 4.8) wereadded, in order, with vortexing 14 seconds after addition of eachreagent, to ensure good mixing.

3) Plates were placed in ice water for 15 minutes.

4) Samples were centrifuged at 3200 rpm for 10 minutes at 4° C.

5) Supernatants were transferred to 96-well Polyfiltronics polypropylenefilterplate (10 micron, 0.8 ml).

6) 500 μl WP resin were added and incubated 3-5 minutes at RT; suctionwas applied to plate.

7) Samples were washed three times with 640 μl of the resuspensionsolution.

8) Samples were centrifuged at 3200 rpm for 5 minutes at RT, to removeresidual buffer.

9) Samples were eluted 2-5 minutes with 40 μl room temperature water.

10) Eluted DNA was centrifuged through to microwell plate at 3200 rpmfor 5 minutes at room temperature.

11) DNA was quantitated.

Pooling Strategy. The pooling strategy was devised to provide optimalsized pools, 1200 cfu, for transfection and detection, and quickbreakdown to the smaller pools of 150. Once a positive pool of 150 wasidentified, between 400 to 800 individual clones were needed to providerepresentation of the pool. Using a single pool of 1200 cfu initiallywould have meant fewer DNA probes but would have required the use ofmore individual clones (3200-6400) in the final identification stepthereby requiring significantly more time to identify a positive clone.

DNAs totalling 5 μg were pooled equally from eight wells, one column, togive a total of 1200 cfu. Thus, each 96-well plate gave rise to 12pooled DNAs for transfection into COS-7 cells.

When a positive pool was identified, DNA was prepared from each of theeight wells constituting the pool and retransfected into COS-7 cells.When a positive well was identified, the well was broken down by platingout an aliquot of the glycerol freeze of that well such that severalthousand individual colonies were obtained. For each positive well,between 400 and 800 colonies were picked and arrayed in a 96-wellformat, DNA was obtained, as described above, and the DNA from 24 wellswas pooled for transfection. DNA representing each individual clone froma positive row was isolated and transfected for final identification.

Quantitative Ob Cell Surface Binding Analysis.

Quantitative cell surface binding assays with AP-Ob fusion proteins wereperformed essentially as described previously for Kit-AP (Flanagan, J.G. and Leder, P., 1990, Cell 63:185-194.

Ob Protein. The recombinant murine Ob protein used herein has beendescribed previously (Campfield et al., 1995, Science 269:546-549). Therecombinant human Ob protein used herein was purified from Baculovirussupernatants with a monoclonal antibody column containing monoclonalantibody directed against human Ob. The purified recombinant human Obprotein was judged by standard Coomasie blue staining to be greater than95% pure.

DNA Sequencing. Sequencing and sequence assembly were performed asdescribed previously (International Polycystic Kidney Consortium, 1995,Cell 81:289-298).

Northern Analysis. Northern blot analysis of poly A⁺ mRNA from varioustissues (Clontech) was probed, using standard techniques (Chirgwin, J.M. et al. 1979, Biochemistry 18:5294-5299), with labeled DNA amplifiedfrom sequences encoding the murine ObR extracellular domain.

rt-PCR. Reverse transcription PCR (rt-PCR) reactions were performed on 1μg total RNA utilizing standard techniques (Zhang, Y. et al., 1994,Nature 372:425-432). Specifically, first strand cDNA was prepared usingrandom hexamers. The first strand cDNA was then PCR amplified usingprimers derived from sequences encoding the ObR extracellular domain orG3PDH control primers.

7.2. RESULTS 7.2.1. CLONING OF THE OB RECEPTOR FROM MOUSE CHOROID PLEXUS

The strong, Ob-specific binding of the AP-Ob fusion protein to themurine choroid plexus described above, in the Example presented inSection 6, suggested that an Ob receptor could be expressed at highlevels within this tissue. In order to attempt to clone a cDNA encodingthe Ob receptor, therefore, the choroid plexuses from 300 mice weredissected, and a total of 11 μg poly A⁺ RNA was isolated from the tissueto be used to construct cDNA libraries as described above, in Section7.1.

Initially, 3 μg poly A⁺ were used to generate cDNA, to be used inconstructing mouse choroid plexus cDNA library A. All of the cDNAsgenerated which were greater than 500 bp in size (261 ng) were pooledand 90 ng were ligated to pMET7. Transformation of this ligated cDNAinto electrocompetent DH10B E. coli resulted in a library ofapproximately 7.2×10⁵ cfu, with an average size of 1 kb.

Recognizing that cDNA library A did not contain a sufficient number ofclones containing inserts large enough to encode a receptor at astatistically reasonable frequency, a second 3 μg of poly A⁺ RNA wasused to generate 758 ng of cDNA. 32 ng of cDNA representing the largesttwo fractions of cDNA were pooled and ligated into pMET7. Transformationof these ligated cDNA molecules resulted in mouse choroid plexus libraryD, with 2.4×10⁵ cfu and an average insert size of 2 kb. Using only thelargest two fractions of cDNA ensured that the library would be biasedtowards large cDNAs. This was confirmed by characterizing the insertsizes of ten clones; seven clones had inserts greater than 2 kb inlength and no clones were seen with inserts smaller than 1 kb. This wasin contrast to the library A where 16 out of 20 clones were smaller than1 kb.

DNA representing 6×10⁵ cfu (40 plates) was prepared and pooled from themouse choroid plexus library A. DNA representing 2.4×10⁵ cfu (16 plates)was prepared from mouse choroid plexus library D.

For screening purposes, the libraries were produced as pools of 150clones, with a mixture of 8 pools being used in each transfection (i.e.,1200 clones/transfection). Pooled DNA was transiently transfected intoCOS-7 cells, and the cells were screened by incubation with supernatantscontaining the murine AP-Ob fusion protein, washed, and stained for APactivity in situ, all as described, above, in Sections 6.1 and 6.2. Oncea positive pool was identified, the 8 individual subpools were eachtested separately, and the resulting positive subpool was furthersubdivided until a single positive clone was identified.

A total of 632 DNA pools were derived from libraries A and D, with atotal of 10 independent positive pools being identified. All of thesepositive pools were successfully broken down into subpools of 150 cloneseach, and one positive subpool was further subdivided until a singlepositive clone was identified. The clone, which contained a 5.1 kB cDNAinsert, was designated famj5312.

7.2.2. THE Ob RECEPTOR (ObR) AND ObR GENE

The famj5312 murine obR cDNA clone isolated, as described above, inSection 7.2.1, contained an insert of approximately 5.1 kb. Thenucleotide sequence obtained from this clone is depicted in FIGS. 1A-1D(SEQ ID NO:1). The nucleotide sequence of the clone revealed a singleopen reading frame, the ObR derived amino acid sequence of which is alsodepicted in FIGS. 1A-1D (SEQ ID NO:2).

The deduced 894 amino acid sequence of the murine ObR protein beginswith a methionine whose codon is within a DNA sequence that isconsistent with a translation initiation site. The ObR amino acidsequence begins with a hydrophobic signal sequence from amino acidresidues 1-23, typical of proteins which are to be eithermembrane-associated or secreted.

The murine Ob receptor protein contains a single hydrophobictransmembrane domain from amino acid residues 838-860, indicating thatthe Ob receptor spans the cell membrane once.

The position of the transmembrane domain indicates that theextracellular portion of the mature murine ObR protein spans from aminoacid residue 24 to amino acid residue 837. A database search revealsthat the extracellular domain of ObR contains regions of homology whichplace ObR into the Class I family of cytokine receptors (for reviews,see, e.g., Heldin, C. -H., 1995, Cell 80:213-223; and Kishimoto, T. andTetsuya, T., 1994, Cell 76:253-252). ObR appears to be most closelyrelated to the gp130 signal transducing component of the IL-6 receptor,the GSF receptor and the LIF receptor. Alignment studies of ObR andgp130 amino acid sequences revealed that, although the overall sequenceidentity between the two proteins is low, the characteristic conservedcysteine residues, the Trp-Ser-X-Trp-Ser motif, and other amino acidresidues conserved within the class I family of proteins are clearlyevident.

Following the single transmembrane domain, the murine Obr proteincontains a short cytoplasmic domain of 34 amino acids (i.e., amino acidresidues 861-894). Homology comparisons also reveal that the firsttwenty three amino acids of the ObR cytoplasmic domain show a 30%identity to membrane proximal sequences of the LIF receptor.

Reverse transcription PCR amplification of obR mRNA from total RNAconfirmed the presence of obR transcript (a single band of about 5 kb)in choroid plexus, and also demonstrated its presence in hypothalamus.Further, Northern blot analysis of poly A⁺ RNA derived from severalmouse tissues revealed that obR mRNA is present in additional tissues,such as lung and kidney.

7.2.3. THE OB RECEPTOR STRONGLY BINDS OB PROTEIN

An analysis of the binding of AP-Ob to the ObR encoded by the obR cDNAdescribed above, in Section 7.2.2, was conducted. The results of thisanalysis, depicted in FIG. 2, demonstrate that the ObR exhibits strong,Ob-specific binding to both mouse and human Ob protein.

A quantitative analysis of the binding of the AP fusion proteins isshown in FIGS. 2A-2B-1 and 2B-2. After transient transfection of the ObRclone into COS cells, strong binding of 1 nM murine AP-Ob is detected(relative to mock transfected COS cells or ObR transfected COS cellsincubated with unfused AP) (FIG. 2A). This binding is nearly completelyinhibited by 100 nM untagged recombinant mouse or human leptin protein,demonstrating that this receptor can bind native Ob. A fusion between APand human Ob also binds mouse ObR with high affinity, as does a fusionprotein with mouse leptin at the N-terminus and AP at the C-terminus(Ob-AP). Scatchard analysis of the binding of mouse AP-Ob FIGS. 2B-1 and2B-2 produced a value for the dissociation constant (KD) of 0.7×10⁻⁹ M.

7.2.4. AUTHENTICITY OF THE famj5312 CLONE

The authenticity of the isolated obR famj5312 clone was tested inseveral ways. First, 8 independently isolated clones (in subpools of 150clones each) were PCR amplified with primers made to obR sequences 3′ ofthe stop codon. Sequencing verified that all 8 clones contained the same3′ untranslated sequences. In addition, the regions of 5 independentlyisolated clones encoding the ObR C-terminus were sequenced and each wasshown to utilize the same stop codon. Finally, reverse transcription PCR(rt-PCR) of choroid plexus total RNA isolated from a different mousestrain (C57/BLKsJ) than that from which the cDNA libraries were derivedgenerated an identical PCR product containing a stop codon in the samelocation. These data indicated that the isolated famj5312 cDNA clone wasneither a chimeric clone nor was it the result of a rare aberrantsplicing event, but, rather, represents a clone which encodes thepredominant form of the ObR receptor in the choroid plexus.

7.2.5. CLONING MOUSE LONG FORM ObR ENCODING NUCLEIC ACIDS

As described herein, we have cloned the murine ObR long form. In orderto find the mouse homolog of the human long form of the obR gene FIGS.3A-3F, semi-nested PCR was performed on first strand cDNA isolated frommouse hypothalamus, Ks, and choroid plexus, db and Ks, with 5′ primersfrom the region just before mouse short form starts to diverge from thehuman long form, and 3′ degenerate primers designed from the human ObRhomolog intracellular region. The complete transcript was furthercharacterized by 3′ RACE.

Total mRNA was prepared from C57B1/KS (KS) and C57B1/KS-db (db) choroidplexus and hypothalamus. cDNA was reverse-transcribed from 1 μg of cDNAof mRNA using random hexamer or oligo dT as primer with SuperscriptReverse Transcriptase from GIBCO-BRL. A total 24 μg of cDNA was made.For PCR, cDNA was diluted 1:200 and 3 μg of the diluted cDNA was used ina 25 μl reaction.

The first round of PCR reactions used a 5′ primer encoding the mouse ObRprotein sequence PNPKNCSW, and consisting of nucleotides 5′-CCA AAC CCCAAG AAT TGT TCC TGG-3′, and a reverse degenerate primer complementary tothe nucleotide sequence encoding KIMENKMCD, adjacent to the carboxyterminus of the human long form and consisting of nucleotides 5′-TC(GA)CA CAT (CT)TT (GA)TT (GATC)CC CAT TAT CTT-3′.

For the second round of PCR reactions, the 3′ primer was the same, andthe 5′ primer, which was internal to the previous 5′ primer, encoded themouse ObR protein sequence AQGLNFQK, and consisted of nucleotides 5′-GCACAA GGA CTG AAT TTC CAA AAG-3′.

PCR reactions were carried out as described above, except the nested PCRprofile was 94° C. for 3 minutes; 94° C. for 30 seconds, 57° for 30seconds, 72° C. for 40 seconds for 30 cycles; 72° C. for 5 minutes forone cycle.

DNA sequencing was performed on the automatic ABI 373A and 377 DNAsequencer by using the Taq cycle sequencing kit (Applied Biosystems,Foster City, Calif.). Sequence analysis was performed using Sequencher.

Semi-nested PCR of the nucleic acids encoding the intracellular domainof murine long form ObR was also performed on mRNA isolated fromhypothalamus in order to obtain sufficient quantities of a specific PCRproduct encoding the mouse long form of obR gene. Sequencing of the PCRproduct FIGS. 6A-6F confirmed that this DNA encodes the mouse homolog ofthe iong form of ObR. The transcripts of the short and long forms areidentical until the fifth codon 5′ of the stop codon of the short formand then diverge completely, suggestive of alternative splicing. Thededuced amino acid sequences from mouse long form and the human ObR arehomologous throughout the length of the coding region and share 75%identity FIGS. 7A-7B.

7.2.6. EXPRESSION PROFILE OF ObR mRNA

As a first step in understanding the tissue distribution of ObR, theexpression of its mRNA was examined in various murine tissues. To thisend, Northern blot analysis of poly A⁺ mRNA (2 μg/lane) derived fromvarious mouse tissues (heart, brain, spleen, lung, liver, skeletalmuscle, kidney and testes; Clontech, Palo Alto, Calif.) was probed withlabelled DNA amplified from sequences encoding the ObR extracellulardomain. Hybridizations were done in Rapid-hyb buffer (Amersham) at 65°C. following the manufacturer's instructions.

In most tissues, the obR mRNA appears as a single band slightly largerthan 5 kb, indicating that the 5.1 kb cDNA clones described herein arefull-length. Of the tissues assayed, expression was seen in lung,kidney, and total brain. No expression was detected in testes.

RT-PCR amplification of the obR mRNA from total RNA confirmed thepresence of this transcript in choroid plexus and also demonstrated itspresence in hypothalamus. The RT-PCR reactions were performed on 1 μgtotal RNA isolated from mouse choroid plexus or hypothalamus. Tissueswere isolated from db/db mice (C57B1/BLKsJ background) or+/+ littermatecontrols. First strand cDNA, prepared using random hexamers, was PCRamplified using primers derived from sequences encoding the ObRextracellular domain or G3PDH control primers. No bands were detectedfrom the amplification of mock reverse-transcribed total RNA controlsrun in parallel.

8. EXAMPLE THE obR GENE IS THE db GENE

The experiments and studies described below demonstrate that the obRgene maps to the db locus, and that the obR gene in db mice is a mutantform of obR that results in transcription of an aberrantly spliced mRNAhaving a 106 nucleotide insert resulting in a truncated long form murineObR protein that is identical to murine short form ObR.

8.1. THE obR GENE MAPS WITHIN THE db GENETIC INTERVAL

In the Example presented herein, studies are described which indicatethat the obR gene maps to a 4 to 5 cM region on mouse chromosome 4 whichrepresents the same region to which the db locus maps.

8.1.1. MATERIALS AND METHODS

PCR Amplification. The following famj5312-derived primers were used foramplification of mouse genomic DNA:

forward primer: 5′-GCTGCACTTAACCTGGC-3′

reverse primer: 5′-GGATAACTCAGGAACG-3′.

The PCR reaction mixture contained 6 μl template DNA (10 ng/μl), 1.4 μl10× Perkin Elmer (Norwalk, Conn.) PCR buffer, 1.12 μl dNTPs (2.5 mM),1.05 μl Forward primer (6.6 μM), 1.05 μl Reverse primer (6.6 μM), 0.38μl H₂O and 3 μl AmpliTaq Hotstart™ polymerase (Perkin Elmer; 0.5 U/μl).

The amplification profile was as follows: 94° C., 2 minutes, at whichpoint the ampliTaq was added, then 30 cycles of 94° C., 40 seconds, 55°C., 50 seconds and 72° C., 30 seconds.

A second set of primers were utilized under the same conditions exceptthat the 55° C. cycle was conducted at 52° C.:

forward primer: 5′-CACTATTTGCCCTTCAG-3′

reverse primer: 5′-GCCTGAGATAGGGGTGC-3′

Electrophoresis. Samples were run on both nondenaturing 8% acrylamidegels run at 45 W, room temperature, for 3 hours and nondenaturing 10%acrylamide SSCP (single stranded conformational polymorphism) gels runat 20 W, 4° C., for 2.5 hours.

Both types of gels were stained with SYBR Green I and scanned on an MDFluorimager, and gave interpretable results.

8.1.2. MAPPING OF THE famj5312 obR cDNA CLONE

PCR primers were designed from the coding sequence of famj5312 cDNA, asdescribed in Section 8.1. These primers amplified a 192 bp fragment fromC57Bl/6J genomic DNA, consistent with the base pair length between thetwo primers in the obR cDNA, and a 195 bp fragment from the wild-typederived Mus spretus strain SPRET/Ei. The 3 bp insertion in the Musspretus allele codes for an additional Asn between amino acids #45 and#46. The genetic segregation of the Mus spretus 195pb allele of ObR wasfollowed in 182 backcross progeny of the cross (C57Bl/6J×Mus spretus) F₁females×C57Bl/6J males by both Single Stranded ConformationalPolymorphism (SSCP) gel electrophesis and nondenaturing gelelectrophoresis for size determination. The segregation pattern of theMus spretus allele was compared to the segregation pattern of 226 othergenetic loci that have been mapped in this backcross panel. Byminimizing the number of multiple crossovers between obR and othermarkers it was determined that obR maps to murine chromosome 4,approximately 2.2±1.6 cM distal to the marker D4Mit9 and 4.6±1.6 cMproximal of the marker D4Mit46. The genetic map position of obR wasfurther refined by mapping additional genetic markers. The obR gene maps0.6±0.6 cM distal from D4Mit255 and 0.6±0.6 cM proximal of D4Mit155; seeFIG. 8.

Additional primer pairs were designed (forward=CACTATTTGCCCTTCAG;reverse=GCCTGAGATAGGGGTGC) from the 3′ sequence of famj5312 cDNA whichalso revealed a polymorphism on SSCP gels between C57Bl/6J genomic DNAand that of the wild derived Mus spretus strain SPRET/Ei. Again thispermitted the genetic mapping of famj5312 cDNA, now using a differentfragment of the clone. The mapping of this polymorphism was 100%concordant with the mapping of famj5312 reported above, both confirmingthe mapping of obR and indicating that the famj5312 cDNA clone was notchimeric.

8.1.3. DEFINITION OF THE MURINE db GENETIC REGION

The mouse db gene was originally mapped to mouse chromosome 4 (Hummel,K.-P. et al., 1966, Science 153:1127-1128). This genetic localizationhas been refined (Bahary, N. et al., 1990, Proc. Natl. Acad. Sci. USA87:8642-8646; Bahary, N. et al., 1993, Genomics 16:113-122) to place dbwithin a genetic interval of 1.5 cM between the proximal Ornithinedecarboxylase 4 (Odc4) locus and the anonymous distal markers D4Rck22and D4Rck69. Bahary et al. 1993 also report D4Mit205 as being 1.1 cMproximal to Odc4. Hence, relative to D4Mit205, the db gene was mappedapproximately 2.2 cM distal.

The db allele originally arose on the C57Bl/BLKsJ inbred strain. The dbmutation has subsequently been transferred to other genetic backgroundsto form congenic strains. By typing animals of the congenic strainC57Bl/6J-m db it was possible to define the genetic interval withinwhich the db gene had to reside on mouse chromosome 4. By this analysis,the interval that must contain the db gene was defined as theapproximate 4 cM between the proximal anonymous DNA marker D4Mit255 andthe distal markers D4Mit331 and D4Mit31. (Genetic distance as defined onthe Mit map; Dietrich, W. F. et al., 1994, Nature Genetics 7:220-245;Copeland, N. G. et al., 1993, Science 262:67; Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995). It should be noted that the interval defined byBahary et al. 1993, supra, appears to be a few centimorgans proximal ofthe region as defined herein. See FIG. 8, in which the distance betweenD4Mit255 and D4Mit31 is about 5.1 cm.

By comparing the mapping data for famj5312 with the db mapping datadescribed above, the map position of famj5312, 0.6±0.6 cM distal fromD4Mit255 and 0.6±0.6 cM proximal of D4Mit155, is in complete accordancewith obR being the db gene.

8.2. THE obR MUTATION IN db MICE RESULTS IN A TRUNCATED LONG FORMRECEPTOR 8.2.1. MATERIALS AND METHODS

Total mRNA was prepared from C57Bl/KS (KS) and C57B1/KS-db (db) choroidplexus and hypothalamus. cDNA was reverse-transcribed from 1 μg of cDNAof mRNA using random hexamer or oligo dT as primer with SuperscriptReverse Transcriptase from GIBCO-BRL. A total 24 μg of cDNA was made.For PCR, cDNA was diluted 1:200 and 3 μg of the diluted cDNA was used ina 25 μl reaction.

From the mouse short form cDNA clone, famj5312, and the long form cDNAclone FIGS. 6A-6F, primers were designed covering the entire codingregion of both the short and long forms of obR cDNA. Overlapping PCRfragments with an average size of 600 bp were generated from eachsample. PCR products were electrophoresed on an 0.8% low melting agarosegel. DNA was isolated from the gel and agarased. Agarased DNA fragmentswere sequenced with both end primers as well as internal primers.

PCR Conditions. The 25 μl PCR reaction contained 2 mM MgCl₂, 0.5 mM ofeach primer, 200 mM each of dATP, dTTP, dCTP and dGTP, and 0.5 units ofTaq polymerase in 1× Taq polymerase buffer (Perkin-Elmer). All PCRreactions were performed in the GeneAmp PCR System 9600 (Perkin-Elmer).Unless otherwise described, the general PCR profile was 94° C. for 3minutes; 94° C. for 10 seconds, 57° C. for 10 seconds, 72° C. for 40seconds for 35 cycles and 72° C. for 5 minutes for one cycle.

DNA sequencing and Sequence Analysis. DNA sequencing was performed onthe automatic ABI 373A and 377 DNA sequencer by using the Taq cyclesequencing kit (Applied Biosystems, Foster City, Calif.). Sequenceanalysis was performed using Sequencher.

8.2.2. RESULTS

Semi-nested PCR was performed on mRNA isolated from choroid plexuses ofKS and db mice. The PCR product generated using the db cDNA as templatewas approximately 100 bp longer than that using Ks DNA as template. ThePCR products from both were directly sequenced. No sequence differencewas detected within the coding sequence of the short form of the mRNAspecies expressed in the choroid plexus of these mice. However, upon thesequencing of the PCR product that was generated starting from thetransmembrane domain shared by the two forms and ending in theintracellular domain specific for the long form, we noticed an apparentdifference between db/db and control in several tissues. The sequencingdata showed that the putative db long form of obR has an additional 106bp insertion in the normal long form transcript (FIG. 9). This 106 bpincludes sequence encoding the last five amino acids, stop codon as wellas 88 bp 3′ UTR region of the short form. The db long form produces atruncated ObR protein identical to the short form which lacks theintracellular domain. We did not detect the normal long form in any dbtissues, nor the db long form in control tissues.

To understand the mechanism of this apparent splicing error, we comparedthe obR genomic sequence between the db/db and control mice. A singlenucleotide change of G→T was discovered 2 bp immediately after the 106bp insertion site in db/db mice. This change creates a splice donorwhich converts the 106 bp fragment to an exon inserted in the db longform. Because of this insertion, the db long form produces only atruncated protein which does not have the intracellular signal domain.Since the class I cytokine receptors to which ObR is most closelyrelated all have a long intercellular domain, the long intercellulardomain of the long form is crucial for initiating intracellular signaltransduction. These data support the role of this receptor in weightmodulation, and the failure to produce ObR long form as cause of thesevere obese phenotype in db/db mice.

9. EXAMPLE: CLONING HUMAN ObR ENCODING NUCLEIC ACIDS

Described herein is the cloning and identification of cDNA and gemonicDNA which encode human obR.

9.1. CLONING THE HUMAN Obr cDNA

The famj5312 cDNA insert was used to probe a human fetal brain cDNAlibrary in the Uni-Zap XR vector obtained from Sratagene (La Jolla,Calif.). A cDNA library derived from a human fetal brain was chosenbecause of the likelihood that this library would contain cDNAs presentin the entire brain, including the choroid plexus, the tissue source ofthe mouse obR cDNA, as well as cDNAs present in the hypothalamus.

The cDNA library was plated on 20 plates with approximately 50,000pfu/plate. Duplicated filter lifts were done on each plate with AmershamHybond-N nylon membrane filters. The filters were denatured, neutralizedand cross-linked according to standard procedures. The probe wasradioactively labelled by random priming in the presence of ³²P labellednucleotide. The filters were hybridized with probe overnight at 65° C.in Church's buffer (7% SDS, 250 mM NaHPO4, 2 μM EDTA, 0.1% BSA). Thenext day, filters were washed in 2×SSC/0.1% SDS for 20 min at 65° C.,then in 0.1×SSC/0.1% SDS for 10 min. They were then exposed to Kodakfilm at −80° C. for 5 hours.

After matching up duplicated filters, 13 duplicated signals wereobtained. Secondary plating was followed by plating out 10 μl of 1:1000dilution of each primary plug. The same probe, hybridization and washconditions were used as above. Film was exposed at 80° C. for 2 hours.Only 1 of the 13 original positives gave duplicated signals on the film.

Four independent plaques from the positive plate were processed andexcised with ExAssist helper phage, XL1-Blue cells and SOLR cells asdescribed by Stratagene. Excision products were then plated out onLB/Amp plates and incubated at 37° C. overnight. One white colony waspicked up from each plate and grown in liquid LB/Amp at 37° C.overnight. The next day mini preps were done with the Promega WizardMini-prep kit. The sizes of the inserts were determined by digesting themini-prep products with EcoRI and XhoI. One of the four clones (d) hasan insert of approximately 6 kb.

DNA for sequencing was prepared using a Qiagen Plasmid Maxi kit.

FIGS 3A-3F depicts the nucleotide sequence (SEQ ID NO:3) of human obRcDNA encoding the signal sequence (amino acid residue 1 to about aminoacid residue 20), extracellular domain (from about amino acid residue 21to about amino acid residue 839), transmembrane domain (from about aminoacid residue 840 to about amino acid residue 862), and cytoplasmicdomain (from about amino acid residue 863 to about amino acid residue1165).

9.2. CLONING HUMAN obR GENOMIC DNA

As described herein, we have cloned human obR genomic DNA.

The famj5312 cDNA insert was used to probe human high density PACfilters purchased from Genome Systems Inc. (Catalog No. FPAC-3386). Theprobe was random prime labelled using the Prime-It kit (Stratagene;Catalog No. 300392). The hybridization was carried out in AmershamRapid-hyb buffer according to the manufacturer's recommendations. Thefilters were then washed in 2×SSC/1% SDS at 65° C. and exposed to Kodakfilm at −80° C.

Eleven putative positive PAC clones were identified. Their grid positionwas determined and the clones were purchased from Genome Systems, Inc.

The clone at grid position P298-K6, which we have designated hobr-p87,was further validated as containing the entire ObR coding region by PCRtesting with primer pairs from the 5′ (obRF4 and obRR4) and 3′ (obRS andobRO) ends of the obR open reading frame. The primers used in thisvalidation were as follows:

obRF4: 5′-CTGCCTGAAGTGTTAGAAGA-3′

obRR4: 5′-GCTGAACTGACATTAGAGGTG-3′

obRS: 5′-ACCTATGAGGACGAAAGCCAGAGAC-3′

obRO: 5′-TGTGAGCAACTGTCCTCGAGAACT-3′

The hobr-p87 clone was deposited with the ATCC on Dec. 28, 1995.

10. EXAMPLE: CONSTRUCTION OF ObR IMMUNOGLOBULIN FUSION PROTEINS 10.1.PREPARATION OF OBR-IG FUSION PROTEINS

The extracellular portion of human ObR is prepared as a fusion proteincoupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications including those whichreduce or eliminate effector activity inherent in the immunoglobulinstructure. (See, e.g., PCT Publication No. WO88/07089, published Sep.,22, 1988). Briefly, PCR overlap extension is applied to join DNAencoding the extracellular portion of human ObR to DNA encoding thehinge, CH2 and CH3 regions of human IgG1. This is accomplished asdescribed in the following subsections.

10.2. PREPARATION OF GENE FUSIONS

PCR reactions are prepared in 100 μl final volume composed of Pfupolymerase and buffer (Stratagene) containing primers (1 μM each), dNTPs(200 μM each), and 1 ng of template DNA.

DNA fragments corresponding to the DNA sequences encoding the ObR ECD,or a portion thereof that binds Ob, are prepared by polymerase chainreaction (PCR) using primer pairs designed so as to amplify sequencesencoding the entire human ObR ECD as well as a small amount of 5′noncoding sequence. For example, the forward primer:5′-GTCACGATGTCGACGTGTACTTCTCTGAAGTAAGATGATTTG-3′ (SEQ ID NO:39)corresponds to nucleotides −20 to +8 in FIGS. 3A-3F with an additional14 nucleotides (containing a SalI site) at the 5′ terminus. The reverseprimer: 5′-GTCAGGTCAGAAAAGCTTATCACTCTGTGTTTTTCAATATCATCTTGAGTGAA-3′ (SEQID NO:40) corresponds to the complement of nucleotides +2482 to +2517 inFIGS. 3A-3F, with an additional 18 nucleotides (containing a HindIIIsite) at the 5′ terminus. A cDNA encoding human ObR serves as thetemplate for amplifying the extracellular domain. PCR amplification withthese primers generates a DNA fragment that encodes ObR extracellulardomain.

In a second PCR reaction, a'second set of primers are designed toamplify the IgG constant region (i.e., the hinge, CH2, and CH3, domains)such that the reverse primer has an unique restriction site and thesequence of the forward primer has a 5′ terminus that is complementaryto the 5′ terminal region of the reverse primer used in the ObR ECDamplification, supra (i.e., 5′-AAGCTTTTCTGACCTGACNNN-3′) and which willenable the open reading frame in the ObR encoding nucleotide sequence tocontinue throughout the length of the IgG nucleotide sequence to beamplified. The template DNA in this reaction is the 2000 nucleotidesegment of human IgG heavy chain genomic DNA (Ellison et al., 1982, Nuc.Acids. Res 10:4071-4079).

The complete human obR-IgG fusion segment is prepared by an additionalPCR reaction. The purified products of the two PCR reactions above aremixed, denatured (95° C., 1 minute) and then renatured (54° C., 30seconds) to allow complementary ends of the two fragments to anneal. Thestrands are filled in using dNTPs and Taq polymerase and the entirefragment amplified using forward PCR primer of the first PCR reactionand the reverse PCR primer of the second PCR reaction. For convenienceof cloning into the expression vector, the resulting fragment is thencleaved with restriction enzymes which recognize unique designed sitesin the forward PCR primer of the first PCR reaction and the reverse PCRprimer of the second PCR reaction. This digested fragment is then clonedinto an expression vector that has also been treated with theserestriction enzymes.

Sequence analysis is used to confirm structure and the construct is usedto transfect COS cells to test transient expression.

Those skilled in the art are aware of various considerations whichinfluence the choice of expression vector into which the obR-IgG fusionsegment is to be cloned, such as the identity of the host organism andthe presence of elements necessary for achieving desired transcriptionaland translational control. For example, if transient expression isdesired, the obR-IgG fusion segment generated supra can be cloned intothe expression vector pcDNA-1 (Invitrogen). Alternatively, stableexpression of the fusion protein can be achieved by cloning the obR-IgGfusion segment into the expression vector pcDNA-3 (Invitrogen).

Alternatively, mouse and/or human obR-IgG fusion proteins can begenerated using an expression vector such as the CD5-IgG1 vector(described by Aruffo et al., 1990, Cell, 61: 1303-1313), which alreadycontains the IgG constant region. According to this method, the DNAfragment encoding the ObR extracellular domain is generated in a PCRreaction so that the open reading frame encoding the ObR extracellulardomain is continuous and in frame with that encoding the IgG constantregion.

For example, the extracellular domains (including signal peptides) ofmouse and human ObR were PCR amplified with Extaq (PanVera Corp.). Thefollowing primers were used for amplification of mouse and human ObR infirst generation expression constructs:

Mouse

Forward primer: 5′-CCCAATGTCGACATGATGTGTCAGAAATTCTAT-3′

Reverse primer: 5′-AAAAAGGATCCGGTCATTCTGCTGCTTGTCGAT-3′

Human

Forward primer: 5′-CCCAATGTCGACATGGTGTACTTCTCTGAAGTA-3′

Reverse primer: 5′-TTTTTGGATCCCACCTGCATCACTCTGGTG-3′

Each forward primer above contains a Sal I restriction site and eachreverse primer above contains a BamHI restriction site. Afteramplification using the mouse and human obR cDNAs as templates, theresulting PCR fragments were cloned into the XhoI/BamHI sites of theCD5-IgG vector (Aruffo et al., 1990, Cell). The resulting vectors weretransiently transfected into COS cells and conditioned media wasgenerated. Immunoprecipitation (IP) of the conditioned media withprotein A and analysis by SDS PAGE revealed that the mouse ObR IgGfusion was expressed at greater levels than human ObR-IgG. To improveexpression of the human ObR-IgG fusion, primers were designed whichamplified the extracellular domain of human ObR (without the signalpeptide) and this fragment was coligated with sequences encoding thesignal-peptide of mouse ObR into the CD5-IgG vector. The followingprimers used for amplification of the human ObR ECD fragment that wasfused with mouse ObR signal peptide:

Forward primer: 5′-TTTAACTTGTCATATCCAATTACTCCTTGGAGATTTAAGTTGTCTTGC-3′

Reverse primer: 5′-TTTTTGGATCCCACCTGCATCACTCTGGTG-3′

After amplification, restriction enzyme digestion, and subcloning, theresulting construct was transiently expressed in COS cells. IP andSDS-PAGE analysis of the resulting conditioned media showed successfulexpression of the 170 KD human ObR IgG fusion. An alternative method forenhancing the expression of immunoglobulin fusion proteins, involvesinsertion of the ObR extracellular domain (not including the signalpeptide) into the CD5-IgG1 vector in such a manner so that the CD5signal peptide is fused to the mature ObR extracellular domain. Such asignal peptide fusion has been shown to improve expression ofimmunoglobulin fusion proteins.

10.3. PREPARATION OF MODIFIED CH2 DOMAINS

The nucleotide sequence of the obR-IgG gene fusion generated supra, canbe modified to replace cysteine residues in the hinge region with serineresidues and/or amino acids within the CH2 domain which are believed tobe required for IgG binding to Fc receptors and complement activation.

Modification of the CH2 domain to replace amino acids thought to beinvolved in binding to Fc receptor is accomplished as follows. Theplasmid construct generated supra, provides the template formodifications of the ObR-IgCλ1 CH2 domain. This template is PCRamplified using the forward PCR primer described in the first PCRreaction supra and a reverse primer designed such that it is homologousto the 5′ terminal portion of the CH2 domain of IgG1 except for fivenucleotide substitutions designed to change amino acids 234, 235, and237 (Canfield, S. M. and Morrison, S. L. (1991) J. Exp. Med.173:1483-1491) from Leu to Ala, Leu to Glu, and Gly to Ala,respectively. Amplification with these PCR primers yields a DNA fragmentconsisting of a modified portion of the CH2 domain. In a second PCRreaction, the template is PCR amplified with the reverse primer used inthe second PCR reaction supra, and a forward primer which is designedsuch that it is complementary to the Ig portion of the molecule andcontains the five complementary nucleotide changes necessary for the CH2amino acid replacements. PCR amplification with these primers yield afragment consisting of the modified portion of the CH2 domain, anintron, the CH3 domain, and 3′ additional sequences. The completeobR-IgCλ1 segment consisting of a modified CH2 domain is prepared by anadditional PCR reaction. The purified products of the two PCR reactionsabove are mixed, denatured (95° C., 1 minute) and then renatured (54°C., 30 seconds) to allow complementary ends of the two fragments toanneal. The strands are filled in using dNTP and Taq polymerase and theentire fragment amplified using forward PCR primer of the first PCRreaction and the reverse PCR primer of the second PCR reaction. Forconvenience of cloning into the expression vector, the resultingfragment is then cleaved with restriction enzymes recognizing sitesspecific to the forward PCR primer of the first PCR reaction and thereverse PCR primer of the second PCR reaction. This digested fragment isthen cloned into an expression vector that has also been treated withthese restriction enzymes.

Sequence analysis is used to confirm structure and the construct is usedto transfect COS cells to test transient expression. hIgG ELISA is usedto measure/confirm transient expression levels approximately equal to100 ng protein/ml cell supernatant for the construct. CHO cell lines aretransfected for permanent expression of the fusion proteins.

10.4. OBR-Ig NEUTRALIZES Ob PROTEIN

To establish whether the ObR-IgG fusion proteins were capable of bindingand neutralizing OB protein (leptin) in vitro and in mice, large scaletransient transfections were performed in 293 cells using the mouseObR-IgG fusion protein. The ObR-IgG protein was purified to nearhomogeneity on a protein A column and analyzed for its ability toinhibit the binding of an alkaline phosphatase-OB fusion protein (AP-OB)to cell surface ObR.

COS cells were transiently transfected with mouse obR cDNA and testedfor their ability to bind 0.5 nM AP-OB. As demonstrated in FIG. 10purified ObR-IgG was able to potently inhibit, or neutralize, thebinding of AP-OB fusion protein to cell surface ObR.

FIG. 10, column 1 shows the high levels of specific binding observed inthe absence of ObR-IgG fusion protein. Columns 2, 3 and 4 show the nearcomplete inhibition of binding observed with three different columnfractions of purified ObR-IgG.

11. THE OBR LONG-FORM HAS SIGNALLING CAPABILITIES OF IL-6 TYPE CYTOKINERECEPTORS

To address whether the cloned ObR isoforms are signaling competent, theObR gene was introduced into established tissue culture cell lines andthe cell response to OB treatment was compared with that mediated by thestructurally-related IL-6 type cytokine receptors. The results presentedin this example provide evidence that the ObR long form is asignal-transducing molecule and shares functional specificity withIL-6-type cytokine receptors.

11.1. MATERIALS AND METHODS 11.1.1 CELLS

COS-1, COS-7, H-35 (Baumann, et al., 1989, Ann. N.Y. Acad. Sci.557:280-297), HepG2, and Hep3B (Lai, et al., 1995, J. Biol. Chem.270:23254-23257) cells were cultured as described. The cells weretreated in medium containing 0.5% fetal calf serum alone or supplementedwith 1 μM dexamethasone, 0.1-1000 ng/ml human OB, 1000 ng/ml mouse OB,IL-6 (Genetics Institute) or G-CSF (Immunex Corp.). To inhibit signalingby gp130, the cells were treated with the combination of twopan-blocking monoclonal antibodies against human gp130, B-R3 (Chevalier,et al., 1995, N.Y. Acad. Sci. 762:482-484) and 144 (20 μg/ml).

11.1.2. EXPRESSION VECTORS AND CAT REPORTER GENE CONSTRUCTS

Expression vectors for the long form of human ObR and the short form ofmouse ObR are described above (Sections 7-9). The truncated humanG-CSFR(27) (Ziegler, et al., 1993, Mol. Cell. Biol., 13:2384-2390) andrat STAT1, STAT3 and STAT5B (Lai, et al., 1995, J. Biol. Chem.,270:23254-23257; Ripperger, et al., 1995, J. Biol. Chem.,270:29998-30006) have been described. ObR with a mutated box 3 sequence(Y1141F) was generated by overlap extension PCR using syntheticoligonucleotides encoding the specified amino acid substitution(Higuchi, et al., 1988, Nucleic Acids Res., 12:5707-5717). The y1141Fcontains a replacement of the tyrosine at position 1141 withphenyalamino. Plasmid SV-SPORT1 (Life Technologies, Inc.) containing ratSTAT3 truncated by 55 carboxy-terminal residues has been generated byconverting codons 716 and 717 to two stop codons. The CAT reporter geneconstructs, pHRRE-CAT and pIL-6RE-CAT, have been described previously(Lia, et al., 1995, J. Biol. Chem., 270:23254-23257; Morella, et al.,1995, J. Biol. Chem., 270:8298-8310).

11.1.3. CELL TRANSFECTION AND ANALYSIS

COS-1, H-35 and Hep3B cells were transfected with plasmid DNA by theDEAE-dextran method (Lopata, et al., 1989, Nucleic Acids Res.,12:5707-5717), HepG2 cells by the calcium phosphate method (Graham, etal., 1973, Virology, 52:456-461), and COS-7 cells by the lipofectaminemethod. Subcultures of COS cells were maintained for 16 hours inserum-free medium prior to the activation of STAT proteins by treatmentwith cytokines for 15 min. DNA binding by STAT proteins were determinedby EMSA on whole cell extracts as described in Sadowski, et al. (1993,Science, 26:1739-1744). Double stranded oligonucleotides for the highaffinity SIEm67 (Sadowski, et al., 1993, Science, 26:1739-1744) and TB-2(Ripperger, et al., 1995, J. Biol. Chem, 270:29998-30006) served as EMSAsubstrates. CAT gene-transfected cell cultures were treated for 24 hourswith cytokines or OB. CAT activities were quantitated by testing serialdilutions of cell extracts, normalized to the expression of thecotransfected marker plasmid pIE-MUP (Morella, et al., 1995, J. Biol.Chem., 270:8298-8310), and are expressed relative to the value of theuntreated control cultures in each experimental series (defined as=1.0).Quantitative cell surface binding of the AP-OB fusion protein (Section6) was done essentially as outlined by Cheng and Flanagan (Cheng &Flanagan, 1994, Cell, 79:157-168).

11.2. RESULTS AND DISCUSSION 11.2.1. OBR ACTIVATES STAT PROTEINS

To determine whether ObR has the ability to recruit the cellularsignaling machinery, COS cells were transiently transfected withexpression vectors for the two representative forms of ObR, mouse shortform (also corresponding to a mutated form detected in db/db mice) andhuman long form. Two days after transfection, cells were incubated in 1nM human or mouse alkaline phosphatase-OB cell surface expression of ObRwas detected as indicated by specific binding of the alkalinephosphatase-OB (AP-OB) fusion protein. Transfection of the short formObR resulted in approximately 10-fold higher binding than the long form.Scatchard transformation of binding data performed at multiple AP-OBconcentrations indicated that the lower binding observed for the longform was mainly a result of reduced cell surface expression. The mouseshort form bound both the murine and human ligands with an affinity of0.7 nM, and the human long form bound both the murine and human ligandswith an affinity of 1.0 nM.

COS-1 cells were co-transfected with expression vectors for human ormouse ObR (2 μg/ml) and the various STAT protein (3 μg/ml).Co-transfection of the expression vectors for ObR and various STATisoforms allowed analysis of the ligand-induced activation of specificSTAT proteins. The transfected cells were treated for 15 min. without orwith murine OB (100 ng/ml) and activation of DNA binding of the STATproteins was identified by EMSA using the diagnostic oligonucleotidesubstrates STE or TB-2. In these experiments, only the long form of ObRactivated either endogenous COS STAT proteins, or the co-expressedSTAT1, STAT3, or STAT5B. Activation of all STAT isoforms by ObR wasligand dependent. In contrast, the short form of ObR was unable toactivate any endogenous or co-transfected STAT proteins despite its highsurface expression. Since the long form of ObR activated all the STATproteins that are also activated by G-CSFR, LIFR and gp130 (Kishimoto,et al., 1995, Blood, 86:1243-1254; Lia, et al., 1995, J. Biol. Chem.270:23254-23257), the long form ObR was predicted to stimulatetranscription with a specificity of the IL-6-type cytokine receptors.

11.2.2. OBR SIGNALS INDUCE GENE EXPRESSION

Rodent and human hepatoma cell lines have previously been utilized todefine the gene-inducing action of ectopically-expressed hematopoietinreceptors (Baumann, et al., Mol. Cell. Biol., 14:138-146). Consequently,three complementary hepatoma cell lines were applied to characterize ObRsignaling. The long or short forms of ObR or human G-CSFR, wereintroduced into rat H-35 cells, together with the HRRE-CAT reporter geneconstruct, the expression of which is increased in these cells bysignals of many hematopoietin receptors (Morella, et al., 1995, J. Biol.Chem., 270:8298-8310). Subcultures were treated for 24 hours withserum-free medium alone or containing cytokines (mOB, LIF or IL-6) withor without dexamethasone. The long form of ObR mediated ligand-dependentinduction of CAT gene expression. The stimulatory action wassynergistically enhanced by dexamethasone. The cell response mediated byObR was highly similar to that of the endogenous IL-6R butcharacteristically different from the endogenous LIFR. In contrast, theshort form of ObR failed to induce gene expression, indicating that the34 residue cytoplasmic domain, despite the presence of a box 1-relatedmotif, was ineffective in recruitment of the cellular signalingcomponents. The fact that the G-CSFR with a cytoplasmic domain truncatedto 27 residues still induced gene transcription in the presence ofligand illustrated that the cells were able to respond to the signalderived from a short, box-1-containing cytoplasmic domain of ahematopoietin receptor. The lack of induction of CAT gene expression inG-CSFR-transfected control cells demonstrates that H-35 cells do notrespond to OB in the absence of transfected ObR.

11.2.3. OBR FUNCTIONS INDEPENDENTLY OF gp130

The results described above support the model that the long form of ObRreconstitutes a signaling pathway similar to that of IL-6R. Next, todetermine whether gp130 is part of the functional ObR, the long form ofObR was introduced together with HRRE-CAT or IL-6RE-CAT into HepG2 cellsand the inhibitory effects of anti gp130 antibodies was assessed.

Treatment of the transfected HepG2 cells with either mouse or human OBproduced a similarly strong induction which was in the range of thatproduced by IL-6 (30-40 fold stimulation). A dose response analysisindicated that maximal regulation was achieved with 100 ng/ml OB. Infour independent experiments, it was established that 1-5 ng/ml OBproduced a half-maximal stimulation, and that 1000 ng/ml yielded astimulation that was consistently below maximum. In the presence ofmonoclonal antibodies against human gp130, which are known to preventsignaling by all IL-6 type cytokine receptors (Chevalier, et al., 1995,N.Y. Acad. Sci. 762:482-484), the stimulation of gene expression by IL-6was abolished as expected, whereas the regulation by OB was unaffected.These results indicate that ObR functions independently of gp130(insensitive to anti-gp130) and that signal initiation may be triggeredby receptor homo-oligomerization.

11.2.4. BOX 3 SEQUENCE OF OBR AND STAT3 ARE INVOLVED IN SIGNALING

Induction of transcription via IL-6 RE is characteristic of thehematopoietin receptors of IL-10R which contain at least one copy of thebox 3 motif (YXXQ) in their cytoplasmic domains (Lai et al., 1995, J.Biol. Chem, 270:23254-23257). This box 3 sequence has been implicated inrecruiting STAT3 to the receptor as part of its activation byreceptor-associated kinases (Lia et al., 1995, J. Biol. Chem.270:23254-23257); Stahl et al., 1995, Science 267:1349-1353). The longform of ObR FIGS. 3A-3F contains at amino acid position 1141 to 1144 onecopy of the box 3 motif that could account for the activation of STAT3and transcriptional stimulation of IL-6RE-CAT. To assess whether the box3 motif of ObR and STAT3 were involved in the gene inducing effect ofObR, two complementary reagents were applied: a box 3-mutant ObR and adominant negative STAT3. The role of box 3-sequence in the long form ofObR was determined by mutating tyrosine at amino acid position 1141 tophenylalanine (Y1141F). Hep G2 and H-35 cells were transfected with anexpression vector for wild-type ObR or ObRY1141F (2 μg/ml) together witheither pHRRE-CAT or pIL-6RE-CAT. Cells were treated with human OB (100ng/ml), and the relative change in CAT activity was determined. Themutant ObR transfected into HepG2 cells yielded a lower stimulation ofboth the HRRE-and IL-6RE-CAT reporter gene constructs than the wild-typeObR. For example, stimulation of HRRE-CAT expression was reduced 40 foldin HepG2 cells and H-35 cells. Stimulation of IL-6RE-CAT was reduced20-fold in HepG2 cells and 100-fold in H-35 cells. Control experimentsindicated that reduced signaling activity of the mutant ObR was not dueto compromised surface expression as shown by AP-OB binding. Therelative effect of the mutation was more prominent on IL-6RE than onHRRE. A similar experiment carried out in H-35 cells showed that box 3mutation was correlated with a loss of IL-6RE regulation, whereas HRREregulation was minimally affected. The results are consistent withprevious observations that, in some cell lines, the recruitment of STAT3was more important in gene induction through IL-6RE then through HRRE(Lai et al., 1995, J Biol. Chem. 270:23254-23257; Morella et al., 1995,J Biol. Chem. 270:8298-8310; Wang et al., 1995, Blood 86:1671-1679).

The reduced gene-regulatory effect of the Y1141F ObR mutant was alsocorrelated with a lower activation of STAT proteins. When the mutant ObRwas transfected into COS-1 cells, as done for the wild-type ObR,activation of the endogenous COS STAT proteins was not detected. Also,ObR Y1141F was approximately 10 times less effective in activatingoverexpressed STAT1 and STAT3 than wild type ObR. Activation of STAT5Bwas, however, unaffected by the mutation. This profile of STATactivation by ObR Y1141F was in agreement with that observed for box3-deficient gp130(Lai, et al., 1995, J. Biol. Chem. 270:23254-23257) andG-CSFR (Morella, et al., 1995, J. Biol. Chem. 270:8298-8310) and wouldexplain the specific changes in the regulation of the reporter geneconstructs.

The signal transducing role of STAT3 was determined by usingover-expression of STAT3⇄55C, a mutant STAT3 with a 55 residue carboxyterminal truncation that acts as dominant negative inhibitor of STAT3action on gene transcription. DNA binding assays such as those describedin Section 11.2.1. , supra, verified that the long form of ObRefficiently activated DNA binding activity of STAT3⇄55C. STAT3⇄55Cessentially abolished the ObR mediated induction of IL-6RE and reducedthat of HRRE by 50π. These data indicate that in the hepatic cells, ObRengages signal transduction pathways that are also utilized by theIL-6-type cytokine receptors and are sensitive to STAT3⇄55C.

11.2.5. OBR CAN UTILIZE BOTH STAT3 AND STAT5B GENE INDUCTION

Induction of the selected reporter gene constructs in HepG2 or H-35cells is maximal and not significantly enhanced by over-expressedwild-type STAT proteins. To assess whether the STAT proteins activatedby ObR play a positive mediator role, human Hep3B cells were transfectedwith human ObR together with either pIL-6RE-CAT or pHRRE-CAT, and theexpression vector for the STAT proteins. Stimulation of CAT activity byhuman OB (100 ng/ml) relative to untreated control was determined(mean±S.D.; N=3 to 4). Those hepatoma cells have retained expression offunctional IL-6R, but lack the receptors to other IL-6-type cytokines(Baumann, et al., 1994, Mol. Cell. Biol. 14:138-146). Moreover, thesecells have a relatively low level of STAT3 and −5, thus permittingtesting of the signaling of ObR by gain of function throughover-expression of STAT proteins. The results from these experimentsindicate that overexpressed STAT3 mediated induction of IL-6RE 15-fold.Overexpressed STAT31 and STAT5B enhanced ObR mediated induction ofHRRE-CAT 5-fold and 30-fold, respectively.

11.3. CONCLUSION

The results presented above documents that full length ObR is a signaltransducing receptor with a mode of action related to the IL-6-typecytokine receptors. The data also support the hypothesis that thetruncated ObR variants, such as the short form expressed in many tissuesor encoded by the db mutant transcript, are either signaling-incompetentor exert a reduced signaling repertoire that is not detectable by thetools applied here. The fact that reconstitution of an OB response isachieved at the level of gene expression in hepatic cells stronglysuggests that an equivalent process may occur in hypothalamic cells orother cell types that normally express the full-length ObR. The link ofObR to specific signaling pathways utilizing STAT proteins and theknowledge of the specificity of these proteins to control genes throughidentifiable DNA binding elements may assist in identifying theimmediate ObR effects that are relevant to understanding OB action invivo. The experimental system presented above can also be used toaddress questions about the functional role, if any, of the naturallyoccurring short forms of ObR in functional regulation of the long form.

12. MUTATIONAL ANALYSIS OF OBR

In order to identify regions of the ObR cytoplasmic region important foractivation of genes, a number of ObR mutants were created and analyzed.These studies, described below, identified two distinct regions of theObR cytoplasmic domain important for induction of gene expression.

12.1 MATERIALS AND METHODS 12.1.1 CELLS

COS-1, COS-7 and H-35 cells were cultured as described by Baumann etal., 1989, Ann. N.Y. Acad. Sci. 557:280-297. Cells were mock stimulatedin medium containing 0.5% fetal calf serum and 1 μM dexamethasone ortreated in the same medium supplemented with 100 ng/ml human leptin(Roche), IL-6 (Genetics Institute), or G-CSF (Immunex Corp.).

12.1.2 EXPRESSION VECTORS AND CAT REPORTER GENE CONSTRUCTS

The expression vectors for the long form of human ObR are describedabove (Section 9) and rat STAT1, STAT3 and STAT5B have been describedpreviously (Lai et al., 1995, J. Biol. Chem. 270:23254-23257; Rippergeret al., 1995, J. Biol. Chem. 270:29998-30006). pOB-RΔ1115-1165,pOB-RΔ1065-1165 and pOB-RΔ965-1165, all encoding carboxy-terminaltruncated human ObRs, were generated by PCR. Briefly, oligonucleotidesspanning the intracellular domain of human ObR were used to generatein-frame stop codons 3′ to the specified amino acids. The PCR fragmentswere digested with EcoRV and XbaI and subcloned into human ObR that hadbeen digested with EcoRV and XbaI. A similar strategy was used togenerate pOB-RΔ868 but with primers generating an MscI-XbaI fragmentthat replaced endogenous human ObR sequences. pOB-RY1141F, encodinghuman ObR with a mutated box 3 sequence was prepared as described inSection 11.1.2. ObR mutants pOB-R(box1mt), containing PNP to SNS changesin the ObR box 1 motif (aa 876 and 878), and mutants pOB-RY986F andpOB-RY1079F, were generated by overlap extension PCR using syntheticoligonucleotides encoding the specified Tyr to Phe amino acidsubstitutions (Higuchi et al., 1988, Nucleic Acids Res. 16:7351-7367).The CAT reporter gene constructs, PHRRE-CAT and pIL-6-CAT have beendescribed previously (Lai et al., 1995, J. Biol. Chem. 270:23254-23257;Morella et al, 1995, J. Biol. Chem. 270:8298-8310).

12.1.3 CELL TRANSFECTION AND ANALYSIS

COS-1 and H-35 cells were transfected by the DEAE-dextran method (Lopataet al., 1984, Nucleic Acids Res. 12:5707-5717), and COS-7 cells by thelipofectamine method (Tartaglia et al., 1995, Cell 83:1263-1271). Foranalysis of STAT protein activation, COS cells were maintained for 16 hin serum-free medium, followed by treatment of cells with 100 ng/mlleptin or G-CSF for 15 min.

For CAT assays, transfected cell cultures were subdivided and treatedwith ligands for 24 hours. CAT reporter activities were determined andare expressed relative to values obtained for untreated control culturesfor each experimental series. DNA binding by STAT proteins was analyzedby electromobility shift assay (EMSA) using whole cell extracts asdescribed by Sadowski et al. (1993, Science 26:1739-1744). Radiolabeleddouble stranded oligonucleotides SIEm67 (for STAT1 and STAT3) and TB-2(for STAT5B) served as binding substrates in the EMSA. Receptorexpression in COS cells was analyzed by quantitative cell surfacebinding of AP-OB fusion protein as described by Cheng and Flanagan(1994, Cell 79:157-168).

12.1.4 IMMUNOBLOTTING

All immunoblotting was done as described by Baumann et al. (1996, Proc.Natl. Acad. Sci. U.S.A. 93: xxx—xxx) and immunoreactive proteins werevisualized by enhanced chemiluminescence detection as described by themanufacturer (Amersham). Rabbit polyclonal antiserum specific for STAT5Bwas from Santa Cruz Biotechnology.

12.2 RESULTS AND DISCUSSION

As discussed above, ObR is a member of the class I cytokine receptorsuperfamily. Receptors of this class lack intrinsic tyrosine kinaseactivity and are activated by ligand-induced receptor homo-dimerizationor hetero-dimerization. In many cases, activation requires activation ofreceptor-associated kinases of the Janus family (JAKs) (Ihle et al.,1994, Trends. Biol. Sci. 19:222-227). JAKs associate with themembrane-proximal domain of the intracellular part of the cytokinereceptors, and serve to initiate signal transduction pathways followingligand induced receptor activation. Included among the downstreamtargets of the JAK proteins are members of the STAT (Signal Transducersand Activators of Transcription) family of transcription factors (Ihleet al., 1994, Trends. Biol. Sci. 19:222-227). The STATs are DNA bindingtranscription factors that contain Src-homology (SH2) domains thatinteract with receptor molecules through phosphorylated tyrosineresidues. STAT proteins are activated by tyrosine phosphorylation, formheterodimers or homodimers, translocate to the nucleus and modulatetranscription of target genes.

12.2.1 THE OBR INTRACELLULAR DOMAIN INCLUDES AT LEAST TWO REGIONSIMPORTANT FOR SIGNALLING

To define regions of the ObR cytoplasmic domain required for signaling,a series of C-terminal deletion mutants were constructed (FIG. 11A).These cDNAs encoding these mutants were transiently co-transfected intoH-35 cells with either IL-6RE-CAT or HRRE-CAT reporter constructs andassayed for their ability to stimulate transcription (FIG. 11B).C-terminal truncations that remove box 3 sequences (aa 1141-1144) of ObRabolish transcriptional activation via IL-6-RE (FIG. 11B; upper panel).This result is consistent with the fact that a Y to F mutation in thesingle box 3 motif or ObR completely disrupts signaling in H-35 cellsvia IL-6RE (Section 11.2.4). In contrast, ObR signaling through HRRE wasminimally reduced by removal of extreme C-terminal sequences and was notcompletely disrupted until removal of the approximately 97 amino acidsbetween 868 and 965 (FIG. 11B).

To insure that the expression vectors for the various ObR mutantsdirected the synthesis of surface localized receptor proteins, COS cellstransfected with each construct were assayed for receptor expression byAP-OB binding studies. C-terminal truncations of ObR generate proteinsthat are expressed at the surface and bind ligand (FIG. 12). Moreover,the expression level of ObR increased with progressive truncation of theintracellular domain.

As discussed above, ObR gene induction via IL-6RE correlates withactivation of STAT1 and STAT3 whereas ObR gene induction via HRRE wasfound to correlate with activation of STAT5B. To further evaluate thecorrelation between HRRE stimulation and STAT5B activation, COS cellswere co-transfected with expression plasmids for STAT5B and the ObRdeletion mutants. Immunoblotting performed on extracts prepared fromthese cells revealed that STAT5B was expressed at relatively equalamounts in each of the transfected cultures. Cells were treated withleptin. EMSA analysis was performed, and STAT protein levels werequantitated by Western blotting. Progressive C-terminal truncations ofObR result in a reduced ability to activate STAT5B and detectable STAT5Bactivation was lost only with removal of the membrane proximal ObRsegment (construct pOBRΔ868-1165). Thus, there appears to be acorrelation between loss of ObR STAT5B activation and gene induction viaHRRE.

To define the relative contribution of the conserved intracellulardomain tyrosine residues and of the membrane proximal box 1 motif tosignaling by ObR via HRRE, mutants OB-RY1141F, OB-RY986F, OB-RY1079F andOB-R(box 1 mt) were generated (FIG. 13A). When analyzed in COS cells,AP-OB binding studies demonstrate that-these mutants are expressed atthe cell surface approximately as well as wild-type ObR. Whentransfected into H-35 cells, OB-RY986F and OB-RY1079F were unchanged intheir ability to regulate HRRE (FIG. 13B). In contrast, mutation of theObR box 1 motif results in a complete loss of regulation of geneinduction through this element. Thus, the box 1 motif of ObR appears tobe an important determining factor for the ability of ObR to activatepathways that can modulate gene induction via HRRE.

Gene induction by ObR through IL-6RE requires sequences near the extremeC-terminus of ObR (FIG. 11B). In contrast, ObR gene induction throughHRRE does not appear to require these C-terminal sequences. Moreover,gene induction via this element is only minimally effected by removal ofObR intracellular domain sequences of approximately 200 amino acidsbetween amino acids 965-1165 but is dependent upon membrane proximalsequences of the approximately 17 amino acids between amino acids 868and 965. Consequently, the proposed box 2 motif of ObR (Lee et al.,1996, Nature 379:632-635) (human ObR aa 1066-1075) does not appear tocontribute to gene induction through HRRE. EMSA analysis suggests geneinduction of HRRE correlates with the ability of ObR to activate STAT5B.Interestingly, OB-RΔ965-1165, which has been deleted of allintracellular domain tyrosine residues and therefore all potential SH2docking sites, is still capable of low-level STAT5B activation andtranscriptional stimulation through HRRE. Only when membrane proximalsequences of ObR are removed (OB-RΔ868-1165), are both HRRE geneinduction and STAT5B activation completely abolished. Consistent withthis, OB-R (box-1 mt), containing a mutated box 1 motif, is similarlyunable to induce gene induction through HRRE and would be predicted tobe unable to activate STAT5B.

13. MULTIMERIZATYION OF OBR

The primary structure of ObR suggests that it is closely related to thesignaling subunits of the IL-6-type cytokine receptors. Members of thisgroup can be activated by either heterodimerization or homodimerization(Kishimoto et al., 1994, Cell 76:253-262; Heldin et al., 1995, Cell80:213-223). Included among the former are the receptors for IL-6,leukemia inhibitory factor (LIF), oncostatin M, IL-11, and ciliaryneurotrophic factor (CNTF), all of which share the common signaltransducer, gp130 (Kishimoto et al., 1994, Cell 76:253-262; Taga et al.,1989, Cell 58:573-581). However, previously we have found that ObRappears to signal independently of gp130 (Baumann et al., 1996, Proc.Natl. Acad. Sci. U.S.A. 93: xxx—xxx. Therefore, ObR may function in thepresence of another accessory chain such as the common signaling subunitutilized by receptors for either IL-3, granulocyte macrophage-colonystimulating factor (GM-CSF) and IL-5 (IL-3Rβ), or IL-2, IL-4, IL-7 andIL-9 (IL-2Rλ). However, ObR signals in hepatoma cells, which do notexpress either IL-3Rβ or IL-Rγ (Wang et al., 1995, Blood 86:1671-1679;Morella et al., 1995, J. Biol. Chem. 270:8298-8310). Alternatively, ObRmay be activated by homodimerization as is found for thegranulocyte-colony stimulating factor receptor (G-CSFR) (Fukanaga etal., 1991, EMBO J. 10:2855-2865; Ishezaka-Ikeda et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:123-127). Therefore, to determine whether ObRhas the ability to dimerize and signal as a homodimer, chimericreceptors encoding the extracellular domain of G-CSFR joined to theintracellular domain of ObR or the reciprocal receptor having theextracellular domain of ObR joined to the intracellular domain of G-CSFRwere constructed and analyzed (FIG. 14A).

13.1 MATERIALS AND METHODS 13.1.1 CELLS

COS-1, COS-7 and H-35 cells were cultured as described by Baumann etal., 1989, Ann. N.Y. Acad. Sci. 557:280-297. Cells were mock stimulatedin medium containing 0.5% fetal calf serum and 1 μM dexamethasone ortreated in the same medium supplemented with 100 ng/ml human leptin(Roche), IL-6 (Genetics Institute), or G-CSF (Immunex Corp.).

13.1.2 EXPRESSION VECTORS AND CAT REPORTER GENE CONSTRUCTS

The expression vectors for the long form of human ObR are describedabove (Section 9), full-length G-CSFR or truncated G-CSFR(Δcyto)(Ziegler et al., 1993, Mol. Cell. Biol. 13:2384-2390), and rat STAT1,STAT3 and STAT5B have been described previously (Lai et al., 1995, J.Biol. Chem. 270:23254-23257; Ripperger et al., 1995, J. Biol. Chem.270:29998-30006). As used herein the term “Δcyto” means deletion of thecytoplasmic domain. The G-CSFR/ObR chimeric. receptor was generated byPCR and encodes the extracellular domain of human G-CSFR (aa 1-598)joined near the transmembrane and intracellular domain of human ObR (aa829-1165). The ObR/G-CSFR chimeric receptor was generated by PCR andencodes the mouse ObR extracellular domain and transmembrane sequences(aa 1-860) joined to the intracellular domain of the human G-CSFR (aa631-813). The CAT reporter gene constructs, pHRRE-CAT and pIL-6-CAT havebeen described previously (Lai et al., 1995, J. Biol. Chem.270:23254-23257; Morella et al, 1995, J. Biol. Chem. 270:8298-8310).

13.1.3 CELL TRANSFECTION AND ANALYSIS

COS-1 and H-35 cells were transfected by the DEAE-dextran method (Lopataet al., 1984, Nucleic Acids Res. 12:5707-5717), and COS-7 cells by thelipofectamine method (Tartaglia et al., 1984, Cell 83:1263-1271). Foranalysis of STAT protein activation, COS cells were maintained for 16hours in serum-free medium, followed by treatment of cells with 100ng/ml leptin or G-CSF for 15 minutes.

For CAT assays, transfected cell cultures were subdivided and treatedwith ligands for 24 hours. CAT reporter activities were determined andare expressed relative to values obtained for untreated control culturesfor each experimental series. DNA binding by STAT proteins was analyzedby electromobility shift assay (EMSA) using whole cell extracts asdescribed by Sadowski et al. (1993, Science 26:1739-1744). Radiolabeleddouble stranded oligonucleotides SIEm67 (for STAT1 and STAT3) and TB-2(for STAT5B) served as binding substrates in the EMSA. Receptorexpression in COS cells was analyzed by quantitative cell surfacebinding of AP-OB fusion protein as described by Cheng and Flanagan(1994, Cell 79:157-168).

13.1.4 IMMUNOBLOTTING

All immunoblotting was done as described by Baumann et al. (1996, Proc.Natl. Acad. Sci. U.S.A. 93: xxx—xxx) and immunoreactive proteins werevisualized by enhanced chemiluminescence detection as described by themanufacturer (Amersham). Rabbit polyclonal antiserum specific for STAT5Bwas from Santa Cruz Biotechnology. Goat polyclonal antiserum againstbacterially expressed extracellular domain of G-CSF-R was prepared atRoswell Park Cancer Institute Springville Laboratories.

13.2 RESULTS AND DISCUSSION

The experiments described below suggest that, while dimerization of theObR cytoplasmic domain may be sufficient for signal transduction, higherorder homo-oligomers can abe formed in response to ligand binding.

13.2.1 HOMODIMERIZATION OBR INTRACELLULAR DOMAINS MAY BE SUFFICIENT FORSIGNAL TRANSDUCTION

Since chimeric receptor complexes have proven quite productive for theanalysis of the mechanism of cytokine receptor activation (Morella etal., 1995, J. Biol. Chem. 270:8298-8310; Vigon et al., 1993, Oncogene8:2607-2615; Baumann et al., 1994, Mol. Cell. Biol. 14:138-146),ObR/G-CSFR and G-CSFR/ObR chimeras were produced and studied as a meansto analyze the mechanism of ObR signaling (FIG. 14A). To analyze whetherthe G-CSFR/ObR chimeric receptor could propagate a ligand induced signalcomparable to that for wild-type ObR, the chimera was tested for STATactivation and for transcriptional stimulation. Co-transfection ofG-CSFR/ObR with STAT proteins yielded a G-CSF-induced activation ofSTAT1, STAT3 and STAT5B. This result is similar to the STAT proteinactivation induced by OB in ObR transfected cells (Section 12).Expression of the chimeric receptor was confirmed by immunoblot analysisof cultures transfected with G-CSFR/ObR. These results suggest thatG-CSF mediated dimerization of ObR cytoplasmic domains can generate anObR-type activation of STAT proteins. In addition, it was found that theG-CSFR/ObR chimera could stimulate transcription as detected bymeasurement of gene induction in H-35 cells following receptorco-transfection with the IL-6RE and HRRE reporter constructs (FIG. 14B).The was response elicited was found to be similar to an induction of thereporter gene constructs by either ObR or endogenous IL-6R.

These results indicate that homodimerization of two ObR cytoplasmicdomains can initiate signaling by ObR, similar to the mechanismmediating signaling by wild-type G-CSFR. However, the G-CSFR/ObR chimeracould not definitively prove that OB ligand has the capability todimerize ObR extracellular domains. Consequently, signaling activity bythe reciprocal chimera containing the ObR extracellular domain joined tothe G-CSFR intracellular domain was analyzed (FIG. 14A). Indeed, theObR/G-CSFR chimera could mediate gene induction comparable to that bywild-type ObR, G-CSFR/ObR and wild-type G-CSFR (FIG. 14B). Thus, takentogether, these results suggest that ObR does not require an accessorychain for signaling and that aggregation of two ObR intracellulardomains appears sufficient for receptor activation.

The fact that aggregation of two ObR intracellular domains is sufficientto generate a signal following ligand-induced activation suggests thatObR may function by receptor homodimerization. If so, signaling by ObRmight be “poisoned” by overexpression of a homodimerizing partner thatis signaling deficient, similar to what has been shown for members ofthe receptor tyrosine kinase family (Paulson et al., 1989, J. Biol.Chem. 264:17615-17618; Svensson et al., 1990, J. Biol. Chem.265:20863-20868; Wen et al., 1992, J. Biol. Chem. 267:2512-2518; Fantlet al., 1993, Annu. Rev. Biochem. 62:453-481). As discussed above(Section 12), ObR containing only the membrane proximal 6 amino acids ofthe cytoplasmic domain is signaling defective (FIG. 11B). Consequently,experiments were performed to determine whether expression of atruncated, signaling deficient ObR could disrupt signaling byfull-length ObR. Cells were co-transfected with increasing amounts oftruncated receptor OB-RΔ868-1165 relative to full-length ObR and theability of these complexes to stimulate expression of a reporter geneconstruct was assayed. Co-transfection of increasing amounts oftruncated ObR does result in decreased signaling by wild-type receptor(FIG. 15A). However, even at a large excess of truncated to full-lengthreceptor, the signaling repression observed did not approach the degreeof reduction observed for repression of G-CSFR signaling byoverexpressed and signaling-deficient truncated G-CSFR(Δcyto) (FIG. 15Aand FIG. 15C). The differing sensitivity to dominant negative repressionobserved for ObR and G-CSFR was a property of their extracellulardomains as shown by dominant negative studies with the receptor chimeras(FIG. 15B and FIG. 15C).

A potential explanation for this weak dominant negative repression ofObR is that interaction of two ObR molecules may require functionaldomains residing in the intracellular region of the receptor. To addressthis possibility, the dominant negative repression of ObR by a mutantreceptor rendered signaling defective by a single amino acidsubstitution (Y1141F) in the ObR box 3 motif was examined. As describedabove, this mutation completely abolished the ability of ObR to modulategene induction via IL-6RE in H-35 cells (Section 12). Consequently, theability of OB-R(Y1141F) to inhibit wild-type ObR signaling via thisenhancer element was investigated. These studies revealed thatincreasing the ratio of transfected mutant OB-RY1141F to wild-typereceptor did not strongly repress signaling (FIG. 15E). Thus, the ObRbox 3 mutant and OB-RΔ868-1165 behave similarly in their ability totrans-repress signaling by wild-type ObR. Interestingly, low levelexpression of either truncated or box 3 mutant ObR receptor generates aslight enhancement of signaling by wild-type ObR. Moreover, a similarpattern was also observed for ObR/G-CSFR signaling in the presence ofincreasing amounts of truncated OB-RΔ868-1165 (FIGS. 15A, 15B and 15C).

As discussed above (Section 11), ObR can signal in hepatoma cells in thepresence of neutralizing antibodies to the gp130 signal transducingcomponent of the IL-6-type cytokine receptors. Moreover, these hepatomado not express the other characterized cytokine receptor accessorychains IL-2Rγ or IL-3Rβ (Wang et al., 1995, Blood 86:1671-1679; Morellaet al., 1995, J. Biol. Chem. 270:8298-8310). Consequently, it ispossible that ObR may function by a mechanism involving receptorhomodimerization. Among members of the class I cytokine receptor family,signaling by the G-CSFR is predicted to be initiated by ligand-inducedreceptor homodimerization (Fukanaga et al., 1991, EMBO J. 10:2855-2865;Ishezaka-Ikeda et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:123-127).As stated above, chimeric receptor complexes have proven quiteproductive for the analysis of the mechanism of cytokine receptoractivation (Morella et al., 1995, supra; Vigon et al., 1993, supra;Baumann et al., 1994, supra), ObR/G-CSFR and G-CSFR/ObR chimeras wereproduced and studied as a means to analyze the mechanism of ObRsignaling. These studies revealed that the G-CSFR/ObR chimera canstrongly activate transcription of both the IL-6RE-CAT and HRRE-CATreporter constructs (FIG. 14B). Since G-CSFR is thought to form ahomodimer when G-CSF is bound, this implies that the aggregation of twointracellular ObR domains is sufficient to initiate receptor signaling.In a similar manner, the ObR/G-CSFR chimera also mediatestranscriptional activation through IL-6RE and HRRE (FIG. 14B). Theseresults show that leptin binding can dimerize two ObR extracellularchains thus inducing the association of at least two intracellularG-CSFR domains and activation of the receptor complex. Moreover, theseresults suggest that it may be possible to generate small molecules orantibodies that act as ObR agonists through simple crosslinking of twoObR chains.

As would be predicted for receptors that are activated by simplehomodimerization, signaling by full length G-CSFR and the G-CSFR/ObRchimera can be greatly diminished by co-expression of a signalingdeficient homodimerizing partner. However, OB-RΔ868-1165 was unable toas efficiently repress signaling by full-length ObR or the ObR/G-CSFRchimera. It is therefore possible that leptin binding to cell surfacereceptors can result in higher-order oligomerization (receptor number>2/complex) as has been shown for IL-10 receptor complexes (Tan et al.,1995, J. Biol. Chem. 21:12906-12911) and for members of theActivin/TGF-βR family (Brand et al., 1993, J. Biol. Chem.268:11500-11503; Weiser et al., 1993, Mol. Cell. Biol. 13:7239-7247;Wrana et al., 1994, Cell 71:1003-1014; Moustakas et al., 1993, J. Biol.Chem. 268:22215-22218; Henis et al., 1994, J. Cell Biol. 126:139-154).According to this model, ligand binding by full-length ObR or ObR/G-CSFRchimera can lead to aggregation of more than two receptor chains, yetjuxtaposition of only two intracellular domains is sufficient for signalgeneration. Such complexes would be predicted to be highly resistant todominant negative repression. The strong repression of signaling byG-CSFR(Δcyto) in complexes containing the G-CSFR/ObR chimerademonstrates that ObR intracellular domain can be efficiently repressedwhen placed in the context of a simple homodimer structure. Although itis possible that OB-RΔ868-1165 localizes to a different region of themembrane than wild-type ObR, it is not likely that mutation of a singletyrosine residue of the ObR intracellular domain (Y1114F) would resultin altered receptor membrane localization. Thus, our observation ofsimilar repression effects mediated by either OB-RΔ868-1165 orOB-RY1141F suggests our results are not due to altered membranelocalization. Low expression levels of either OB-RΔ868-1165 andOB-RY1141F generate a small enhancement of. signaling for full lengthObR and the ObR/G-CSFR chimera. We speculate that this effect isattributable to either ligand presentation (Andres et al., 1989, J. CellBiol. 109:3137-3145; Massaugue et al., 1992, Cell 69:1067-1070; Lin etal., 1993, Trends. Cell Biol. 3:14-19) or ligand passing as haspreviously been observed for the TNF receptor (Tartaglia et al., 1993,J. Biol. Chem. 268:18542-18548).

As noted above, it is possible that the short forms of ObR serve atransport or clearance function in the body (Tartaglia et al., 1995,Cell 83:1263-1271). However, the possiblity that the long and shortforms of ObR can functionally interact raises suggests that the shortform of ObR could regulate activities of the long form. This issupported by the fact that the major naturally occurring non-signallingshort form of ObR in the mouse (containing a 34 amino acid intracellulardomain), which also corresponds to the mutant ObR found in the db/dbmouse, can repress long form receptor signaling.

14. DEPOSIT OF MICROORGANISMS

The following microorganism was deposited with the American Type CultureCollection (ATCC), Rockville, Md., on the dates indicated and wereassigned the indicated accession number:

ATTC Date Microorganism Clone Access. No. of Deposit E. coli strainfamj5312 69952 November 22, 1995 5312B4F3 E. coli h-ObRD fahj5312d 69963December 5, 1995 E. coli h-ObR-p87 h-ObR-p87 69972 December 28, 1995

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

50 3097 base pairs nucleic acid double unknown cDNA Coding Sequence61...2742 1 GTCGACCCAC GCGTCCGGAG GAATCGTTCT GCAAATCCAG GTGTACACCTCTGAAGAAAG 60 ATG ATG TGT CAG AAA TTC TAT GTG GTT TTG TTA CAC TGG GAATTT CTT 108 Met Met Cys Gln Lys Phe Tyr Val Val Leu Leu His Trp Glu PheLeu 1 5 10 15 TAT GTG ATA GCT GCA CTT AAC CTG GCA TAT CCA ATC TCT CCCTGG AAA 156 Tyr Val Ile Ala Ala Leu Asn Leu Ala Tyr Pro Ile Ser Pro TrpLys 20 25 30 TTT AAG TTG TTT TGT GGA CCA CCG AAC ACA ACC GAT GAC TCC TTTCTC 204 Phe Lys Leu Phe Cys Gly Pro Pro Asn Thr Thr Asp Asp Ser Phe Leu35 40 45 TCA CCT GCT GGA GCC CCA AAC AAT GCC TCG GCT TTG AAG GGG GCT TCT252 Ser Pro Ala Gly Ala Pro Asn Asn Ala Ser Ala Leu Lys Gly Ala Ser 5055 60 GAA GCA ATT GTT GAA GCT AAA TTT AAT TCA AGT GGT ATC TAC GTT CCT300 Glu Ala Ile Val Glu Ala Lys Phe Asn Ser Ser Gly Ile Tyr Val Pro 6570 75 80 GAG TTA TCC AAA ACA GTC TTC CAC TGT TGC TTT GGG AAT GAG CAA GGT348 Glu Leu Ser Lys Thr Val Phe His Cys Cys Phe Gly Asn Glu Gln Gly 8590 95 CAA AAC TGC TCT GCA CTC ACA GAC AAC ACT GAA GGG AAG ACA CTG GCT396 Gln Asn Cys Ser Ala Leu Thr Asp Asn Thr Glu Gly Lys Thr Leu Ala 100105 110 TCA GTA GTG AAG GCT TCA GTT TTT CGC CAG CTA GGT GTA AAC TGG GAC444 Ser Val Val Lys Ala Ser Val Phe Arg Gln Leu Gly Val Asn Trp Asp 115120 125 ATA GAG TGC TGG ATG AAA GGG GAC TTG ACA TTA TTC ATC TGT CAT ATG492 Ile Glu Cys Trp Met Lys Gly Asp Leu Thr Leu Phe Ile Cys His Met 130135 140 GAG CCA TTA CCT AAG AAC CCC TTC AAG AAT TAT GAC TCT AAG GTC CAT540 Glu Pro Leu Pro Lys Asn Pro Phe Lys Asn Tyr Asp Ser Lys Val His 145150 155 160 CTT TTA TAT GAT CTG CCT GAA GTC ATA GAT GAT TCG CCT CTG CCCCCA 588 Leu Leu Tyr Asp Leu Pro Glu Val Ile Asp Asp Ser Pro Leu Pro Pro165 170 175 CTG AAA GAC AGC TTT CAG ACT GTC CAA TGC AAC TGC AGT CTT CGGGGA 636 Leu Lys Asp Ser Phe Gln Thr Val Gln Cys Asn Cys Ser Leu Arg Gly180 185 190 TGT GAA TGT CAT GTG CCG GTA CCC AGA GCC AAA CTC AAC TAC GCTCTT 684 Cys Glu Cys His Val Pro Val Pro Arg Ala Lys Leu Asn Tyr Ala Leu195 200 205 CTG ATG TAT TTG GAA ATC ACA TCT GCC GGT GTG AGT TTT CAG TCACCT 732 Leu Met Tyr Leu Glu Ile Thr Ser Ala Gly Val Ser Phe Gln Ser Pro210 215 220 CTG ATG TCA CTG CAG CCC ATG CTT GTT GTG AAA CCC GAT CCA CCCTTA 780 Leu Met Ser Leu Gln Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu225 230 235 240 GGT TTG CAT ATG GAA GTC ACA GAT GAT GGT AAT TTA AAG ATTTCT TGG 828 Gly Leu His Met Glu Val Thr Asp Asp Gly Asn Leu Lys Ile SerTrp 245 250 255 GAC AGC CAA ACA ATG GCA CCA TTT CCG CTT CAA TAT CAG GTGAAA TAT 876 Asp Ser Gln Thr Met Ala Pro Phe Pro Leu Gln Tyr Gln Val LysTyr 260 265 270 TTA GAG AAT TCT ACA ATT GTA AGA GAG GCT GCT GAA ATT GTCTCA GCT 924 Leu Glu Asn Ser Thr Ile Val Arg Glu Ala Ala Glu Ile Val SerAla 275 280 285 ACA TCT CTG CTG GTA GAC AGT GTG CTT CCT GGA TCT TCA TATGAG GTC 972 Thr Ser Leu Leu Val Asp Ser Val Leu Pro Gly Ser Ser Tyr GluVal 290 295 300 CAG GTG AGG AGC AAG AGA CTG GAT GGT TCA GGA GTC TGG AGTGAC TGG 1020 Gln Val Arg Ser Lys Arg Leu Asp Gly Ser Gly Val Trp Ser AspTrp 305 310 315 320 AGT TCA CCT CAA GTC TTT ACC ACA CAA GAT GTT GTG TATTTT CCA CCC 1068 Ser Ser Pro Gln Val Phe Thr Thr Gln Asp Val Val Tyr PhePro Pro 325 330 335 AAA ATT CTG ACT AGT GTT GGA TCG AAT GCT TCT TTT CATTGC ATC TAC 1116 Lys Ile Leu Thr Ser Val Gly Ser Asn Ala Ser Phe His CysIle Tyr 340 345 350 AAA AAC GAA AAC CAG ATT ATC TCC TCA AAA CAG ATA GTTTGG TGG AGG 1164 Lys Asn Glu Asn Gln Ile Ile Ser Ser Lys Gln Ile Val TrpTrp Arg 355 360 365 AAT CTA GCT GAG AAA ATC CCT GAG ATA CAG TAC AGC ATTGTG AGT GAC 1212 Asn Leu Ala Glu Lys Ile Pro Glu Ile Gln Tyr Ser Ile ValSer Asp 370 375 380 CGA GTT AGC AAA GTT ACC TTC TCC AAC CTG AAA GCC ACCAGA CCT CGA 1260 Arg Val Ser Lys Val Thr Phe Ser Asn Leu Lys Ala Thr ArgPro Arg 385 390 395 400 GGG AAG TTT ACC TAT GAC GCA GTG TAC TGC TGC AATGAG CAG GCG TGC 1308 Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn GluGln Ala Cys 405 410 415 CAT CAC CGC TAT GCT GAA TTA TAC GTG ATC GAT GTCAAT ATC AAT ATA 1356 His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp Val AsnIle Asn Ile 420 425 430 TCA TGT GAA ACT GAC GGG TAC TTA ACT AAA ATG ACTTGC AGA TGG TCA 1404 Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys Met Thr CysArg Trp Ser 435 440 445 CCC AGC ACA ATC CAA TCA CTA GTG GGA AGC ACT GTGCAG CTG AGG TAT 1452 Pro Ser Thr Ile Gln Ser Leu Val Gly Ser Thr Val GlnLeu Arg Tyr 450 455 460 CAC AGG CGC AGC CTG TAT TGT CCT GAT AGT CCA TCTATT CAT CCT ACG 1500 His Arg Arg Ser Leu Tyr Cys Pro Asp Ser Pro Ser IleHis Pro Thr 465 470 475 480 TCT GAG CCC AAA AAC TGC GTC TTA CAG AGA GACGGC TTT TAT GAA TGT 1548 Ser Glu Pro Lys Asn Cys Val Leu Gln Arg Asp GlyPhe Tyr Glu Cys 485 490 495 GTT TTC CAG CCA ATC TTT CTA TTA TCT GGC TATACA ATG TGG ATC AGG 1596 Val Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr ThrMet Trp Ile Arg 500 505 510 ATC AAC CAT TCT TTA GGT TCA CTT GAC TCG CCACCA ACG TGT GTC CTT 1644 Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro ProThr Cys Val Leu 515 520 525 CCT GAC TCC GTA GTA AAA CCA CTA CCT CCA TCTAAC GTA AAA GCA GAG 1692 Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser AsnVal Lys Ala Glu 530 535 540 ATT ACT GTA AAC ACT GGA TTA TTG AAA GTA TCTTGG GAA AAG CCA GTC 1740 Ile Thr Val Asn Thr Gly Leu Leu Lys Val Ser TrpGlu Lys Pro Val 545 550 555 560 TTT CCG GAG AAT AAC CTT CAA TTC CAG ATTCGA TAT GGC TTA AGT GGA 1788 Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile ArgTyr Gly Leu Ser Gly 565 570 575 AAA GAA ATA CAA TGG AAG ACA CAT GAG GTATTC GAT GCA AAG TCA AAG 1836 Lys Glu Ile Gln Trp Lys Thr His Glu Val PheAsp Ala Lys Ser Lys 580 585 590 TCT GCC AGC CTG CTG GTG TCA GAC CTC TGTGCA GTC TAT GTG GTC CAG 1884 Ser Ala Ser Leu Leu Val Ser Asp Leu Cys AlaVal Tyr Val Val Gln 595 600 605 GTT CGC TGC CGG CGG TTG GAT GGA CTA GGATAT TGG AGT AAT TGG AGC 1932 Val Arg Cys Arg Arg Leu Asp Gly Leu Gly TyrTrp Ser Asn Trp Ser 610 615 620 AGT CCA GCC TAT ACG CTT GTC ATG GAT GTAAAA GTT CCT ATG AGA GGG 1980 Ser Pro Ala Tyr Thr Leu Val Met Asp Val LysVal Pro Met Arg Gly 625 630 635 640 CCT GAA TTT TGG AGA AAA ATG GAT GGGGAC GTT ACT AAA AAG GAG AGA 2028 Pro Glu Phe Trp Arg Lys Met Asp Gly AspVal Thr Lys Lys Glu Arg 645 650 655 AAT GTC ACC TTG CTT TGG AAG CCC CTGACG AAA AAT GAC TCA CTG TGT 2076 Asn Val Thr Leu Leu Trp Lys Pro Leu ThrLys Asn Asp Ser Leu Cys 660 665 670 AGT GTG AGG AGG TAC GTT GTG AAG CATCGT ACT GCC CAC AAT GGG ACG 2124 Ser Val Arg Arg Tyr Val Val Lys His ArgThr Ala His Asn Gly Thr 675 680 685 TGG TCA GAA GAT GTG GGA AAT CGG ACCAAT CTC ACT TTC CTG TGG ACA 2172 Trp Ser Glu Asp Val Gly Asn Arg Thr AsnLeu Thr Phe Leu Trp Thr 690 695 700 GAA CCA GCG CAC ACT GTT ACA GTT CTGGCT GTC AAT TCC CTC GGC GCT 2220 Glu Pro Ala His Thr Val Thr Val Leu AlaVal Asn Ser Leu Gly Ala 705 710 715 720 TCC CTT GTG AAT TTT AAC CTT ACCTTC TCA TGG CCC ATG AGT AAA GTG 2268 Ser Leu Val Asn Phe Asn Leu Thr PheSer Trp Pro Met Ser Lys Val 725 730 735 AGT GCT GTG GAG TCA CTC AGT GCTTAT CCC CTG AGC AGC AGC TGT GTC 2316 Ser Ala Val Glu Ser Leu Ser Ala TyrPro Leu Ser Ser Ser Cys Val 740 745 750 ATC CTT TCC TGG ACA CTG TCA CCTGAT GAT TAT AGT CTG TTA TAT CTG 2364 Ile Leu Ser Trp Thr Leu Ser Pro AspAsp Tyr Ser Leu Leu Tyr Leu 755 760 765 GTT ATT GAA TGG AAG ATC CTT AATGAA GAT GAT GGA ATG AAG TGG CTT 2412 Val Ile Glu Trp Lys Ile Leu Asn GluAsp Asp Gly Met Lys Trp Leu 770 775 780 AGA ATT CCC TCG AAT GTT AAA AAGTTT TAT ATC CAC GAT AAT TTT ATT 2460 Arg Ile Pro Ser Asn Val Lys Lys PheTyr Ile His Asp Asn Phe Ile 785 790 795 800 CCC ATC GAG AAA TAT CAG TTTAGT CTT TAC CCA GTA TTT ATG GAA GGA 2508 Pro Ile Glu Lys Tyr Gln Phe SerLeu Tyr Pro Val Phe Met Glu Gly 805 810 815 GTT GGA AAA CCA AAG ATA ATTAAT GGT TTC ACC AAA GAT GCT ATC GAC 2556 Val Gly Lys Pro Lys Ile Ile AsnGly Phe Thr Lys Asp Ala Ile Asp 820 825 830 AAG CAG CAG AAT GAC GCA GGGCTG TAT GTC ATT GTA CCC ATA ATT ATT 2604 Lys Gln Gln Asn Asp Ala Gly LeuTyr Val Ile Val Pro Ile Ile Ile 835 840 845 TCC TCT TGT GTC CTA CTG CTCGGA ACA CTG TTA ATT TCA CAC CAG AGA 2652 Ser Ser Cys Val Leu Leu Leu GlyThr Leu Leu Ile Ser His Gln Arg 850 855 860 ATG AAA AAG TTG TTT TGG GACGAT GTT CCA AAC CCC AAG AAT TGT TCC 2700 Met Lys Lys Leu Phe Trp Asp AspVal Pro Asn Pro Lys Asn Cys Ser 865 870 875 880 TGG GCA CAA GGA CTG AATTTC CAA AAG AGA ACG GAC ACT CTT 2742 Trp Ala Gln Gly Leu Asn Phe Gln LysArg Thr Asp Thr Leu 885 890 TGAAGTCTCT CATGACCACT ACAGATGAAC CCAATCTACCAACTTCCCAA CAGTCCATAC 2802 AATATTAGAA GATGTTTACA TTTTGATGGA GGGAAACAAACCTAAACTAT GGTTTGAATG 2862 ACTAAGAAAT AACATTTGAT GAGCTTATTA GAGAAGTGTATATTTTGTGG CCACAATGTA 2922 GGTTTGATGT AGTTCAGTTT GGGACATATG CTTGATTTTCAGGGCATCAA AAATTTAAAG 2982 TTGATATTCA TGGACTCTGC ATTTTATTTC TTAAGTCATAAAATGATAAT GGTGTGACGG 3042 TTGGTGTCAG AACCTATTTG GGTACAGATC ACCAAAATATGGTAGGTAAT GCCTT 3097 894 amino acids amino acid unknown proteininternal 2 Met Met Cys Gln Lys Phe Tyr Val Val Leu Leu His Trp Glu PheLeu 1 5 10 15 Tyr Val Ile Ala Ala Leu Asn Leu Ala Tyr Pro Ile Ser ProTrp Lys 20 25 30 Phe Lys Leu Phe Cys Gly Pro Pro Asn Thr Thr Asp Asp SerPhe Leu 35 40 45 Ser Pro Ala Gly Ala Pro Asn Asn Ala Ser Ala Leu Lys GlyAla Ser 50 55 60 Glu Ala Ile Val Glu Ala Lys Phe Asn Ser Ser Gly Ile TyrVal Pro 65 70 75 80 Glu Leu Ser Lys Thr Val Phe His Cys Cys Phe Gly AsnGlu Gln Gly 85 90 95 Gln Asn Cys Ser Ala Leu Thr Asp Asn Thr Glu Gly LysThr Leu Ala 100 105 110 Ser Val Val Lys Ala Ser Val Phe Arg Gln Leu GlyVal Asn Trp Asp 115 120 125 Ile Glu Cys Trp Met Lys Gly Asp Leu Thr LeuPhe Ile Cys His Met 130 135 140 Glu Pro Leu Pro Lys Asn Pro Phe Lys AsnTyr Asp Ser Lys Val His 145 150 155 160 Leu Leu Tyr Asp Leu Pro Glu ValIle Asp Asp Ser Pro Leu Pro Pro 165 170 175 Leu Lys Asp Ser Phe Gln ThrVal Gln Cys Asn Cys Ser Leu Arg Gly 180 185 190 Cys Glu Cys His Val ProVal Pro Arg Ala Lys Leu Asn Tyr Ala Leu 195 200 205 Leu Met Tyr Leu GluIle Thr Ser Ala Gly Val Ser Phe Gln Ser Pro 210 215 220 Leu Met Ser LeuGln Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu 225 230 235 240 Gly LeuHis Met Glu Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp 245 250 255 AspSer Gln Thr Met Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr 260 265 270Leu Glu Asn Ser Thr Ile Val Arg Glu Ala Ala Glu Ile Val Ser Ala 275 280285 Thr Ser Leu Leu Val Asp Ser Val Leu Pro Gly Ser Ser Tyr Glu Val 290295 300 Gln Val Arg Ser Lys Arg Leu Asp Gly Ser Gly Val Trp Ser Asp Trp305 310 315 320 Ser Ser Pro Gln Val Phe Thr Thr Gln Asp Val Val Tyr PhePro Pro 325 330 335 Lys Ile Leu Thr Ser Val Gly Ser Asn Ala Ser Phe HisCys Ile Tyr 340 345 350 Lys Asn Glu Asn Gln Ile Ile Ser Ser Lys Gln IleVal Trp Trp Arg 355 360 365 Asn Leu Ala Glu Lys Ile Pro Glu Ile Gln TyrSer Ile Val Ser Asp 370 375 380 Arg Val Ser Lys Val Thr Phe Ser Asn LeuLys Ala Thr Arg Pro Arg 385 390 395 400 Gly Lys Phe Thr Tyr Asp Ala ValTyr Cys Cys Asn Glu Gln Ala Cys 405 410 415 His His Arg Tyr Ala Glu LeuTyr Val Ile Asp Val Asn Ile Asn Ile 420 425 430 Ser Cys Glu Thr Asp GlyTyr Leu Thr Lys Met Thr Cys Arg Trp Ser 435 440 445 Pro Ser Thr Ile GlnSer Leu Val Gly Ser Thr Val Gln Leu Arg Tyr 450 455 460 His Arg Arg SerLeu Tyr Cys Pro Asp Ser Pro Ser Ile His Pro Thr 465 470 475 480 Ser GluPro Lys Asn Cys Val Leu Gln Arg Asp Gly Phe Tyr Glu Cys 485 490 495 ValPhe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg 500 505 510Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu 515 520525 Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Asn Val Lys Ala Glu 530535 540 Ile Thr Val Asn Thr Gly Leu Leu Lys Val Ser Trp Glu Lys Pro Val545 550 555 560 Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly LeuSer Gly 565 570 575 Lys Glu Ile Gln Trp Lys Thr His Glu Val Phe Asp AlaLys Ser Lys 580 585 590 Ser Ala Ser Leu Leu Val Ser Asp Leu Cys Ala ValTyr Val Val Gln 595 600 605 Val Arg Cys Arg Arg Leu Asp Gly Leu Gly TyrTrp Ser Asn Trp Ser 610 615 620 Ser Pro Ala Tyr Thr Leu Val Met Asp ValLys Val Pro Met Arg Gly 625 630 635 640 Pro Glu Phe Trp Arg Lys Met AspGly Asp Val Thr Lys Lys Glu Arg 645 650 655 Asn Val Thr Leu Leu Trp LysPro Leu Thr Lys Asn Asp Ser Leu Cys 660 665 670 Ser Val Arg Arg Tyr ValVal Lys His Arg Thr Ala His Asn Gly Thr 675 680 685 Trp Ser Glu Asp ValGly Asn Arg Thr Asn Leu Thr Phe Leu Trp Thr 690 695 700 Glu Pro Ala HisThr Val Thr Val Leu Ala Val Asn Ser Leu Gly Ala 705 710 715 720 Ser LeuVal Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val 725 730 735 SerAla Val Glu Ser Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val 740 745 750Ile Leu Ser Trp Thr Leu Ser Pro Asp Asp Tyr Ser Leu Leu Tyr Leu 755 760765 Val Ile Glu Trp Lys Ile Leu Asn Glu Asp Asp Gly Met Lys Trp Leu 770775 780 Arg Ile Pro Ser Asn Val Lys Lys Phe Tyr Ile His Asp Asn Phe Ile785 790 795 800 Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Val Phe MetGlu Gly 805 810 815 Val Gly Lys Pro Lys Ile Ile Asn Gly Phe Thr Lys AspAla Ile Asp 820 825 830 Lys Gln Gln Asn Asp Ala Gly Leu Tyr Val Ile ValPro Ile Ile Ile 835 840 845 Ser Ser Cys Val Leu Leu Leu Gly Thr Leu LeuIle Ser His Gln Arg 850 855 860 Met Lys Lys Leu Phe Trp Asp Asp Val ProAsn Pro Lys Asn Cys Ser 865 870 875 880 Trp Ala Gln Gly Leu Asn Phe GlnLys Arg Thr Asp Thr Leu 885 890 3871 base pairs nucleic acid doubleunknown cDNA Coding Sequence 194...3688 3 GGCACGAGCC GGTCTGGCTTGGGCAGGCTG CCCGGGCCGT GGCAGGAAGC CGGAAGCAGC 60 CGCGGCCCCA GTTCGGGAGACATGGCGGGC GTTAAAGCTC TCGTGGCATT ATCCTTCAGT 120 GGGGCTATTG GACTGACTTTTCTTATGCTG GGATGTGCCT TAGAGGATTA TGGGTGTACT 180 TCTCTGAAGT AAG ATG ATTTGT CAA AAA TTC TGT GTG GTT TTG TTA CAT 229 Met Ile Cys Gln Lys Phe CysVal Val Leu Leu His 1 5 10 TGG GAA TTT ATT TAT GTG ATA ACT GCG TTT AACTTG TCA TAT CCA ATT 277 Trp Glu Phe Ile Tyr Val Ile Thr Ala Phe Asn LeuSer Tyr Pro Ile 15 20 25 ACT CCT TGG AGA TTT AAG TTG TCT TGC ATG CCA CCAAAT TCA ACC TAT 325 Thr Pro Trp Arg Phe Lys Leu Ser Cys Met Pro Pro AsnSer Thr Tyr 30 35 40 GAC TAC TTC CTT TTG CCT GCT GGA CTC TCA AAG AAT ACTTCA AAT TCG 373 Asp Tyr Phe Leu Leu Pro Ala Gly Leu Ser Lys Asn Thr SerAsn Ser 45 50 55 60 AAT GGA CAT TAT GAG ACA GCT GTT GAA CCT AAG TTT AATTCA AGT GGT 421 Asn Gly His Tyr Glu Thr Ala Val Glu Pro Lys Phe Asn SerSer Gly 65 70 75 ACT CAC TTT TCT AAC TTA TCC AAA ACA ACT TTC CAC TGT TGCTTT CGG 469 Thr His Phe Ser Asn Leu Ser Lys Thr Thr Phe His Cys Cys PheArg 80 85 90 AGT GAG CAA GAT AGA AAC TGC TCC TTA TGT GCA GAC AAC ATT GAAGGA 517 Ser Glu Gln Asp Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly95 100 105 AAG ACA TTT GTT TCA ACA GTA AAT TCT TTA GTT TTT CAA CAA ATAGAT 565 Lys Thr Phe Val Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile Asp110 115 120 GCA AAC TGG AAC ATA CAG TGC TGG CTA AAA GGA GAC TTA AAA TTATTC 613 Ala Asn Trp Asn Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe125 130 135 140 ATC TGT TAT GTG GAG TCA TTA TTT AAG AAT CTA TTC AGG AATTAT AAC 661 Ile Cys Tyr Val Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn TyrAsn 145 150 155 TAT AAG GTC CAT CTT TTA TAT GTT CTG CCT GAA GTG TTA GAAGAT TCA 709 Tyr Lys Val His Leu Leu Tyr Val Leu Pro Glu Val Leu Glu AspSer 160 165 170 CCT CTG GTT CCC CAA AAA GGC AGT TTT CAG ATG GTT CAC TGCAAT TGC 757 Pro Leu Val Pro Gln Lys Gly Ser Phe Gln Met Val His Cys AsnCys 175 180 185 AGT GTT CAT GAA TGT TGT GAA TGT CTT GTG CCT GTG CCA ACAGCC AAA 805 Ser Val His Glu Cys Cys Glu Cys Leu Val Pro Val Pro Thr AlaLys 190 195 200 CTC AAC GAC ACT CTC CTT ATG TGT TTG AAA ATC ACA TCT GGTGGA GTA 853 Leu Asn Asp Thr Leu Leu Met Cys Leu Lys Ile Thr Ser Gly GlyVal 205 210 215 220 ATT TTC CAG TCA CCT CTA ATG TCA GTT CAG CCC ATA AATATG GTG AAG 901 Ile Phe Gln Ser Pro Leu Met Ser Val Gln Pro Ile Asn MetVal Lys 225 230 235 CCT GAT CCA CCA TTA GGT TTG CAT ATG GAA ATC ACA GATGAT GGT AAT 949 Pro Asp Pro Pro Leu Gly Leu His Met Glu Ile Thr Asp AspGly Asn 240 245 250 TTA AAG ATT TCT TGG TCC AGC CCA CCA TTG GTA CCA TTTCCA CTT CAA 997 Leu Lys Ile Ser Trp Ser Ser Pro Pro Leu Val Pro Phe ProLeu Gln 255 260 265 TAT CAA GTG AAA TAT TCA GAG AAT TCT ACA ACA GTT ATCAGA GAA GCT 1045 Tyr Gln Val Lys Tyr Ser Glu Asn Ser Thr Thr Val Ile ArgGlu Ala 270 275 280 GAC AAG ATT GTC TCA GCT ACA TCC CTG CTA GTA GAC AGTATA CTT CCT 1093 Asp Lys Ile Val Ser Ala Thr Ser Leu Leu Val Asp Ser IleLeu Pro 285 290 295 300 GGG TCT TCG TAT GAG GTT CAG GTG AGG GGC AAG AGACTG GAT GGC CCA 1141 Gly Ser Ser Tyr Glu Val Gln Val Arg Gly Lys Arg LeuAsp Gly Pro 305 310 315 GGA ATC TGG AGT GAC TGG AGT ACT CCT CGT GTC TTTACC ACA CAA GAT 1189 Gly Ile Trp Ser Asp Trp Ser Thr Pro Arg Val Phe ThrThr Gln Asp 320 325 330 GTC ATA TAC TTT CCA CCT AAA ATT CTG ACA AGT GTTGGG TCT AAT GTT 1237 Val Ile Tyr Phe Pro Pro Lys Ile Leu Thr Ser Val GlySer Asn Val 335 340 345 TCT TTT CAC TGC ATC TAT AAG AAG GAA AAC AAG ATTGTT CCC TCA AAA 1285 Ser Phe His Cys Ile Tyr Lys Lys Glu Asn Lys Ile ValPro Ser Lys 350 355 360 GAG ATT GTT TGG TGG ATG AAT TTA GCT GAG AAA ATTCCT CAA AGC CAG 1333 Glu Ile Val Trp Trp Met Asn Leu Ala Glu Lys Ile ProGln Ser Gln 365 370 375 380 TAT GAT GTT GTG AGT GAT CAT GTT AGC AAA GTTACT TTT TTC AAT CTG 1381 Tyr Asp Val Val Ser Asp His Val Ser Lys Val ThrPhe Phe Asn Leu 385 390 395 AAT GAA ACC AAA CCT CGA GGA AAG TTT ACC TATGAT GCA GTG TAC TGC 1429 Asn Glu Thr Lys Pro Arg Gly Lys Phe Thr Tyr AspAla Val Tyr Cys 400 405 410 TGC AAT GAA CAT GAA TGC CAT CAT CGC TAT GCTGAA TTA TAT GTG ATT 1477 Cys Asn Glu His Glu Cys His His Arg Tyr Ala GluLeu Tyr Val Ile 415 420 425 GAT GTC AAT ATC AAT ATC TCA TGT GAA ACT GATGGG TAC TTA ACT AAA 1525 Asp Val Asn Ile Asn Ile Ser Cys Glu Thr Asp GlyTyr Leu Thr Lys 430 435 440 ATG ACT TGC AGA TGG TCA ACC AGT ACA ATC CAGTCA CTT GCG GAA AGC 1573 Met Thr Cys Arg Trp Ser Thr Ser Thr Ile Gln SerLeu Ala Glu Ser 445 450 455 460 ACT TTG CAA TTG AGG TAT CAT AGG AGC AGCCTT TAC TGT TCT GAT ATT 1621 Thr Leu Gln Leu Arg Tyr His Arg Ser Ser LeuTyr Cys Ser Asp Ile 465 470 475 CCA TCT ATT CAT CCC ATA TCT GAG CCC AAAGAT TGC TAT TTG CAG AGT 1669 Pro Ser Ile His Pro Ile Ser Glu Pro Lys AspCys Tyr Leu Gln Ser 480 485 490 GAT GGT TTT TAT GAA TGC ATT TTC CAG CCAATC TTC CTA TTA TCT GGC 1717 Asp Gly Phe Tyr Glu Cys Ile Phe Gln Pro IlePhe Leu Leu Ser Gly 495 500 505 TAC ACA ATG TGG ATT AGG ATC AAT CAC TCTCTA GGT TCA CTT GAC TCT 1765 Tyr Thr Met Trp Ile Arg Ile Asn His Ser LeuGly Ser Leu Asp Ser 510 515 520 CCA CCA ACA TGT GTC CTT CCT GAT TCT GTGGTG AAG CCA CTG CCT CCA 1813 Pro Pro Thr Cys Val Leu Pro Asp Ser Val ValLys Pro Leu Pro Pro 525 530 535 540 TCC AGT GTG AAA GCA GAA ATT ACT ATAAAC ATT GGA TTA TTG AAA ATA 1861 Ser Ser Val Lys Ala Glu Ile Thr Ile AsnIle Gly Leu Leu Lys Ile 545 550 555 TCT TGG GAA AAG CCA GTC TTT CCA GAGAAT AAC CTT CAA TTC CAG ATT 1909 Ser Trp Glu Lys Pro Val Phe Pro Glu AsnAsn Leu Gln Phe Gln Ile 560 565 570 CGC TAT GGT TTA AGT GGA AAA GAA GTACAA TGG AAG ATG TAT GAG GTT 1957 Arg Tyr Gly Leu Ser Gly Lys Glu Val GlnTrp Lys Met Tyr Glu Val 575 580 585 TAT GAT GCA AAA TCA AAA TCT GTC AGTCTC CCA GTT CCA GAC TTG TGT 2005 Tyr Asp Ala Lys Ser Lys Ser Val Ser LeuPro Val Pro Asp Leu Cys 590 595 600 GCA GTC TAT GCT GTT CAG GTG CGC TGTAAG AGG CTA GAT GGA CTG GGA 2053 Ala Val Tyr Ala Val Gln Val Arg Cys LysArg Leu Asp Gly Leu Gly 605 610 615 620 TAT TGG AGT AAT TGG AGC AAT CCAGCC TAC ACA GTT GTC ATG GAT ATA 2101 Tyr Trp Ser Asn Trp Ser Asn Pro AlaTyr Thr Val Val Met Asp Ile 625 630 635 AAA GTT CCT ATG AGA GGA CCT GAATTT TGG AGA ATA ATT AAT GGA GAT 2149 Lys Val Pro Met Arg Gly Pro Glu PheTrp Arg Ile Ile Asn Gly Asp 640 645 650 ACT ATG AAA AAG GAG AAA AAT GTCACT TTA CTT TGG AAG CCC CTG ATG 2197 Thr Met Lys Lys Glu Lys Asn Val ThrLeu Leu Trp Lys Pro Leu Met 655 660 665 AAA AAT GAC TCA TTG TGC AGT GTTCAG AGA TAT GTG ATA AAC CAT CAT 2245 Lys Asn Asp Ser Leu Cys Ser Val GlnArg Tyr Val Ile Asn His His 670 675 680 ACT TCC TGC AAT GGA ACA TGG TCAGAA GAT GTG GGA AAT CAC ACG AAA 2293 Thr Ser Cys Asn Gly Thr Trp Ser GluAsp Val Gly Asn His Thr Lys 685 690 695 700 TTC ACT TTC CTG TGG ACA GAGCAA GCA CAT ACT GTT ACG GTT CTG GCC 2341 Phe Thr Phe Leu Trp Thr Glu GlnAla His Thr Val Thr Val Leu Ala 705 710 715 ATC AAT TCA ATT GGT GCT TCTGTT GCA AAT TTT AAT TTA ACC TTT TCA 2389 Ile Asn Ser Ile Gly Ala Ser ValAla Asn Phe Asn Leu Thr Phe Ser 720 725 730 TGG CCT ATG AGC AAA GTA AATATC GTG CAG TCA CTC AGT GCT TAT CCT 2437 Trp Pro Met Ser Lys Val Asn IleVal Gln Ser Leu Ser Ala Tyr Pro 735 740 745 TTA AAC AGC AGT TGT GTG ATTGTT TCC TGG ATA CTA TCA CCC AGT GAT 2485 Leu Asn Ser Ser Cys Val Ile ValSer Trp Ile Leu Ser Pro Ser Asp 750 755 760 TAC AAG CTA ATG TAT TTT ATTATT GAG TGG AAA AAT CTT AAT GAA GAT 2533 Tyr Lys Leu Met Tyr Phe Ile IleGlu Trp Lys Asn Leu Asn Glu Asp 765 770 775 780 GGT GAA ATA AAA TGG CTTAGA ATC TCT TCA TCT GTT AAG AAG TAT TAT 2581 Gly Glu Ile Lys Trp Leu ArgIle Ser Ser Ser Val Lys Lys Tyr Tyr 785 790 795 ATC CAT GAT CAT TTT ATCCCC ATT GAG AAG TAC CAG TTC AGT CTT TAC 2629 Ile His Asp His Phe Ile ProIle Glu Lys Tyr Gln Phe Ser Leu Tyr 800 805 810 CCA ATA TTT ATG GAA GGAGTG GGA AAA CCA AAG ATA ATT AAT AGT TTC 2677 Pro Ile Phe Met Glu Gly ValGly Lys Pro Lys Ile Ile Asn Ser Phe 815 820 825 ACT CAA GAT GAT ATT GAAAAA CAC CAG AGT GAT GCA GGT TTA TAT GTA 2725 Thr Gln Asp Asp Ile Glu LysHis Gln Ser Asp Ala Gly Leu Tyr Val 830 835 840 ATT GTG CCA GTA ATT ATTTCC TCT TCC ATC TTA TTG CTT GGA ACA TTA 2773 Ile Val Pro Val Ile Ile SerSer Ser Ile Leu Leu Leu Gly Thr Leu 845 850 855 860 TTA ATA TCA CAC CAAAGA ATG AAA AAG CTA TTT TGG GAA GAT GTT CCG 2821 Leu Ile Ser His Gln ArgMet Lys Lys Leu Phe Trp Glu Asp Val Pro 865 870 875 AAC CCC AAG AAT TGTTCC TGG GCA CAA GGA CTT AAT TTT CAG AAG CCA 2869 Asn Pro Lys Asn Cys SerTrp Ala Gln Gly Leu Asn Phe Gln Lys Pro 880 885 890 GAA ACG TTT GAG CATCTT TTT ATC AAG CAT ACA GCA TCA GTG ACA TGT 2917 Glu Thr Phe Glu His LeuPhe Ile Lys His Thr Ala Ser Val Thr Cys 895 900 905 GGT CCT CTT CTT TTGGAG CCT GAA ACA ATT TCA GAA GAT ATC AGT GTT 2965 Gly Pro Leu Leu Leu GluPro Glu Thr Ile Ser Glu Asp Ile Ser Val 910 915 920 GAT ACA TCA TGG AAAAAT AAA GAT GAG ATG ATG CCA ACA ACT GTG GTC 3013 Asp Thr Ser Trp Lys AsnLys Asp Glu Met Met Pro Thr Thr Val Val 925 930 935 940 TCT CTA CTT TCAACA ACA GAT CTT GAA AAG GGT TCT GTT TGT ATT AGT 3061 Ser Leu Leu Ser ThrThr Asp Leu Glu Lys Gly Ser Val Cys Ile Ser 945 950 955 GAC CAG TTC AACAGT GTT AAC TTC TCT GAG GCT GAG GGT ACT GAG GTA 3109 Asp Gln Phe Asn SerVal Asn Phe Ser Glu Ala Glu Gly Thr Glu Val 960 965 970 ACC TAT GAG GACGAA AGC CAG AGA CAA CCC TTT GTT AAA TAC GCC ACG 3157 Thr Tyr Glu Asp GluSer Gln Arg Gln Pro Phe Val Lys Tyr Ala Thr 975 980 985 CTG ATC AGC AACTCT AAA CCA AGT GAA ACT GGT GAA GAA CAA GGG CTT 3205 Leu Ile Ser Asn SerLys Pro Ser Glu Thr Gly Glu Glu Gln Gly Leu 990 995 1000 ATA AAT AGT TCAGTC ACC AAG TGC TTC TCT AGC AAA AAT TCT CCG TTG 3253 Ile Asn Ser Ser ValThr Lys Cys Phe Ser Ser Lys Asn Ser Pro Leu 1005 1010 1015 1020 AAG GATTCT TTC TCT AAT AGC TCA TGG GAG ATA GAG GCC CAG GCA TTT 3301 Lys Asp SerPhe Ser Asn Ser Ser Trp Glu Ile Glu Ala Gln Ala Phe 1025 1030 1035 TTTATA TTA TCA GAT CAG CAT CCC AAC ATA ATT TCA CCA CAC CTC ACA 3349 Phe IleLeu Ser Asp Gln His Pro Asn Ile Ile Ser Pro His Leu Thr 1040 1045 1050TTC TCA GAA GGA TTG GAT GAA CTT TTG AAA TTG GAG GGA AAT TTC CCT 3397 PheSer Glu Gly Leu Asp Glu Leu Leu Lys Leu Glu Gly Asn Phe Pro 1055 10601065 GAA GAA AAT AAT GAT AAA AAG TCT ATC TAT TAT TTA GGG GTC ACC TCA3445 Glu Glu Asn Asn Asp Lys Lys Ser Ile Tyr Tyr Leu Gly Val Thr Ser1070 1075 1080 ATC AAA AAG AGA GAG AGT GGT GTG CTT TTG ACT GAC AAG TCAAGG GTA 3493 Ile Lys Lys Arg Glu Ser Gly Val Leu Leu Thr Asp Lys Ser ArgVal 1085 1090 1095 1100 TCG TGC CCA TTC CCA GCC CCC TGT TTA TTC ACG GACATC AGA GTT CTC 3541 Ser Cys Pro Phe Pro Ala Pro Cys Leu Phe Thr Asp IleArg Val Leu 1105 1110 1115 CAG GAC AGT TGC TCA CAC TTT GTA GAA AAT AATATC AAC TTA GGA ACT 3589 Gln Asp Ser Cys Ser His Phe Val Glu Asn Asn IleAsn Leu Gly Thr 1120 1125 1130 TCT AGT AAG AAG ACT TTT GCA TCT TAC ATGCCT CAA TTC CAA ACT TGT 3637 Ser Ser Lys Lys Thr Phe Ala Ser Tyr Met ProGln Phe Gln Thr Cys 1135 1140 1145 TCT ACT CAG ACT CAT AAG ATC ATG GAAAAC AAG ATG TGT GAC CTA ACT 3685 Ser Thr Gln Thr His Lys Ile Met Glu AsnLys Met Cys Asp Leu Thr 1150 1155 1160 GTG TAATTTCACT GAAGAAACCTTCAGATTTGT GTTATAATGG GTAATATAAA 3738 Val 1165 GTGTAATAGA TTATAGTTGTGGGTGGGAGA GAGAAAAGAA ACCAGAGTCC AAATTTGAAA 3798 ATAATTGTTC CCAACTGAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 3858 AAAAAAAAAA AAA 38711165 amino acids amino acid unknown protein internal 4 Met Ile Cys GlnLys Phe Cys Val Val Leu Leu His Trp Glu Phe Ile 1 5 10 15 Tyr Val IleThr Ala Phe Asn Leu Ser Tyr Pro Ile Thr Pro Trp Arg 20 25 30 Phe Lys LeuSer Cys Met Pro Pro Asn Ser Thr Tyr Asp Tyr Phe Leu 35 40 45 Leu Pro AlaGly Leu Ser Lys Asn Thr Ser Asn Ser Asn Gly His Tyr 50 55 60 Glu Thr AlaVal Glu Pro Lys Phe Asn Ser Ser Gly Thr His Phe Ser 65 70 75 80 Asn LeuSer Lys Thr Thr Phe His Cys Cys Phe Arg Ser Glu Gln Asp 85 90 95 Arg AsnCys Ser Leu Cys Ala Asp Asn Ile Glu Gly Lys Thr Phe Val 100 105 110 SerThr Val Asn Ser Leu Val Phe Gln Gln Ile Asp Ala Asn Trp Asn 115 120 125Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys Leu Phe Ile Cys Tyr Val 130 135140 Glu Ser Leu Phe Lys Asn Leu Phe Arg Asn Tyr Asn Tyr Lys Val His 145150 155 160 Leu Leu Tyr Val Leu Pro Glu Val Leu Glu Asp Ser Pro Leu ValPro 165 170 175 Gln Lys Gly Ser Phe Gln Met Val His Cys Asn Cys Ser ValHis Glu 180 185 190 Cys Cys Glu Cys Leu Val Pro Val Pro Thr Ala Lys LeuAsn Asp Thr 195 200 205 Leu Leu Met Cys Leu Lys Ile Thr Ser Gly Gly ValIle Phe Gln Ser 210 215 220 Pro Leu Met Ser Val Gln Pro Ile Asn Met ValLys Pro Asp Pro Pro 225 230 235 240 Leu Gly Leu His Met Glu Ile Thr AspAsp Gly Asn Leu Lys Ile Ser 245 250 255 Trp Ser Ser Pro Pro Leu Val ProPhe Pro Leu Gln Tyr Gln Val Lys 260 265 270 Tyr Ser Glu Asn Ser Thr ThrVal Ile Arg Glu Ala Asp Lys Ile Val 275 280 285 Ser Ala Thr Ser Leu LeuVal Asp Ser Ile Leu Pro Gly Ser Ser Tyr 290 295 300 Glu Val Gln Val ArgGly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser 305 310 315 320 Asp Trp SerThr Pro Arg Val Phe Thr Thr Gln Asp Val Ile Tyr Phe 325 330 335 Pro ProLys Ile Leu Thr Ser Val Gly Ser Asn Val Ser Phe His Cys 340 345 350 IleTyr Lys Lys Glu Asn Lys Ile Val Pro Ser Lys Glu Ile Val Trp 355 360 365Trp Met Asn Leu Ala Glu Lys Ile Pro Gln Ser Gln Tyr Asp Val Val 370 375380 Ser Asp His Val Ser Lys Val Thr Phe Phe Asn Leu Asn Glu Thr Lys 385390 395 400 Pro Arg Gly Lys Phe Thr Tyr Asp Ala Val Tyr Cys Cys Asn GluHis 405 410 415 Glu Cys His His Arg Tyr Ala Glu Leu Tyr Val Ile Asp ValAsn Ile 420 425 430 Asn Ile Ser Cys Glu Thr Asp Gly Tyr Leu Thr Lys MetThr Cys Arg 435 440 445 Trp Ser Thr Ser Thr Ile Gln Ser Leu Ala Glu SerThr Leu Gln Leu 450 455 460 Arg Tyr His Arg Ser Ser Leu Tyr Cys Ser AspIle Pro Ser Ile His 465 470 475 480 Pro Ile Ser Glu Pro Lys Asp Cys TyrLeu Gln Ser Asp Gly Phe Tyr 485 490 495 Glu Cys Ile Phe Gln Pro Ile PheLeu Leu Ser Gly Tyr Thr Met Trp 500 505 510 Ile Arg Ile Asn His Ser LeuGly Ser Leu Asp Ser Pro Pro Thr Cys 515 520 525 Val Leu Pro Asp Ser ValVal Lys Pro Leu Pro Pro Ser Ser Val Lys 530 535 540 Ala Glu Ile Thr IleAsn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys 545 550 555 560 Pro Val PhePro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu 565 570 575 Ser GlyLys Glu Val Gln Trp Lys Met Tyr Glu Val Tyr Asp Ala Lys 580 585 590 SerLys Ser Val Ser Leu Pro Val Pro Asp Leu Cys Ala Val Tyr Ala 595 600 605Val Gln Val Arg Cys Lys Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn 610 615620 Trp Ser Asn Pro Ala Tyr Thr Val Val Met Asp Ile Lys Val Pro Met 625630 635 640 Arg Gly Pro Glu Phe Trp Arg Ile Ile Asn Gly Asp Thr Met LysLys 645 650 655 Glu Lys Asn Val Thr Leu Leu Trp Lys Pro Leu Met Lys AsnAsp Ser 660 665 670 Leu Cys Ser Val Gln Arg Tyr Val Ile Asn His His ThrSer Cys Asn 675 680 685 Gly Thr Trp Ser Glu Asp Val Gly Asn His Thr LysPhe Thr Phe Leu 690 695 700 Trp Thr Glu Gln Ala His Thr Val Thr Val LeuAla Ile Asn Ser Ile 705 710 715 720 Gly Ala Ser Val Ala Asn Phe Asn LeuThr Phe Ser Trp Pro Met Ser 725 730 735 Lys Val Asn Ile Val Gln Ser LeuSer Ala Tyr Pro Leu Asn Ser Ser 740 745 750 Cys Val Ile Val Ser Trp IleLeu Ser Pro Ser Asp Tyr Lys Leu Met 755 760 765 Tyr Phe Ile Ile Glu TrpLys Asn Leu Asn Glu Asp Gly Glu Ile Lys 770 775 780 Trp Leu Arg Ile SerSer Ser Val Lys Lys Tyr Tyr Ile His Asp His 785 790 795 800 Phe Ile ProIle Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Ile Phe Met 805 810 815 Glu GlyVal Gly Lys Pro Lys Ile Ile Asn Ser Phe Thr Gln Asp Asp 820 825 830 IleGlu Lys His Gln Ser Asp Ala Gly Leu Tyr Val Ile Val Pro Val 835 840 845Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly Thr Leu Leu Ile Ser His 850 855860 Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro Lys Asn 865870 875 880 Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Pro Glu Thr PheGlu 885 890 895 His Leu Phe Ile Lys His Thr Ala Ser Val Thr Cys Gly ProLeu Leu 900 905 910 Leu Glu Pro Glu Thr Ile Ser Glu Asp Ile Ser Val AspThr Ser Trp 915 920 925 Lys Asn Lys Asp Glu Met Met Pro Thr Thr Val ValSer Leu Leu Ser 930 935 940 Thr Thr Asp Leu Glu Lys Gly Ser Val Cys IleSer Asp Gln Phe Asn 945 950 955 960 Ser Val Asn Phe Ser Glu Ala Glu GlyThr Glu Val Thr Tyr Glu Asp 965 970 975 Glu Ser Gln Arg Gln Pro Phe ValLys Tyr Ala Thr Leu Ile Ser Asn 980 985 990 Ser Lys Pro Ser Glu Thr GlyGlu Glu Gln Gly Leu Ile Asn Ser Ser 995 1000 1005 Val Thr Lys Cys PheSer Ser Lys Asn Ser Pro Leu Lys Asp Ser Phe 1010 1015 1020 Ser Asn SerSer Trp Glu Ile Glu Ala Gln Ala Phe Phe Ile Leu Ser 025 1030 1035 1040Asp Gln His Pro Asn Ile Ile Ser Pro His Leu Thr Phe Ser Glu Gly 10451050 1055 Leu Asp Glu Leu Leu Lys Leu Glu Gly Asn Phe Pro Glu Glu AsnAsn 1060 1065 1070 Asp Lys Lys Ser Ile Tyr Tyr Leu Gly Val Thr Ser IleLys Lys Arg 1075 1080 1085 Glu Ser Gly Val Leu Leu Thr Asp Lys Ser ArgVal Ser Cys Pro Phe 1090 1095 1100 Pro Ala Pro Cys Leu Phe Thr Asp IleArg Val Leu Gln Asp Ser Cys 105 1110 1115 1120 Ser His Phe Val Glu AsnAsn Ile Asn Leu Gly Thr Ser Ser Lys Lys 1125 1130 1135 Thr Phe Ala SerTyr Met Pro Gln Phe Gln Thr Cys Ser Thr Gln Thr 1140 1145 1150 His LysIle Met Glu Asn Lys Met Cys Asp Leu Thr Val 1155 1160 1165 488 aminoacids amino acid unknown protein 5 Tyr Ile Ser Pro Glu Ser Pro Val ValGln Leu His Ser Asn Phe Thr 1 5 10 15 Ala Val Cys Val Leu Lys Glu LysCys Met Asp Tyr Phe His Val Asn 20 25 30 Ala Asn Tyr Ile Val Trp Lys ThrAsn His Phe Thr Ile Pro Lys Glu 35 40 45 Gln Tyr Thr Ile Ile Asn Arg ThrAla Ser Ser Val Thr Phe Thr Asp 50 55 60 Ile Ala Ser Leu Asn Ile Gln LeuThr Cys Asn Ile Leu Thr Phe Gly 65 70 75 80 Gln Leu Glu Gln Asn Val TyrGly Ile Thr Ile Ile Ser Gly Leu Pro 85 90 95 Pro Glu Lys Pro Lys Asn LeuSer Cys Ile Val Asn Glu Gly Lys Lys 100 105 110 Met Arg Cys Glu Trp AspGly Gly Arg Glu Thr His Leu Glu Thr Asn 115 120 125 Phe Thr Leu Lys SerGlu Trp Ala Thr His Lys Phe Ala Asp Cys Lys 130 135 140 Ala Lys Arg AspThr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val 145 150 155 160 Tyr PheVal Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly 165 170 175 LysVal Thr Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys 180 185 190Pro Asn Pro Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser 195 200205 Ser Ile Leu Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile 210215 220 Ile Leu Lys Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp225 230 235 240 Ser Gln Ile Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser SerPhe Thr 245 250 255 Val Gln Asp Leu Lys Pro Phe Thr Glu Tyr Val Phe ArgIle Arg Cys 260 265 270 Met Lys Glu Asp Gly Lys Gly Tyr Trp Ser Asp TrpSer Glu Glu Ala 275 280 285 Ser Gly Ile Thr Tyr Glu Asp Arg Pro Ser LysAla Pro Ser Phe Trp 290 295 300 Tyr Lys Ile Asp Pro Ser His Thr Gln GlyTyr Arg Thr Val Gln Leu 305 310 315 320 Val Trp Lys Thr Leu Pro Pro PheGlu Ala Asn Gly Lys Ile Leu Asp 325 330 335 Tyr Glu Val Thr Leu Thr ArgTrp Lys Ser His Leu Gln Asn Tyr Thr 340 345 350 Val Asn Ala Thr Lys LeuThr Val Asn Leu Thr Asn Asp Arg Tyr Leu 355 360 365 Ala Thr Leu Thr ValArg Asn Leu Val Gly Lys Ser Asp Ala Ala Val 370 375 380 Leu Thr Ile ProAla Cys Asp Phe Gln Ala Thr His Pro Val Met Asp 385 390 395 400 Leu LysAla Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr 405 410 415 ProArg Glu Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser 420 425 430Asp Lys Ala Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val 435 440445 His Arg Thr Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu 450455 460 Ile Thr Val Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser465 470 475 480 Ile Lys Ala Tyr Leu Lys Gln Ala 485 5 amino acids aminoacid unknown peptide 6 Trp Ser Xaa Trp Ser 1 5 17 base pairs nucleicacid single linear DNA 7 CATCTTACTT CAGAGAA 17 23 base pairs nucleicacid single linear DNA 8 CATCTTACTT CAGAGAAGTA CAC 23 29 base pairsnucleic acid single linear DNA 9 CATCTTACTT CAGAGAAGTA CACCCATAA 29 35base pairs nucleic acid single linear DNA 10 CATCTTACTT CAGAGAAGTACACCCATAAT CCTCT 35 35 base pairs nucleic acid single linear DNA 11AATCATCTTA CTTCAGAGAA GTACACCCAT AATCC 35 29 base pairs nucleic acidsingle linear DNA 12 CTTACTTCAG AGAAGTACAC CCATAATCC 29 23 base pairsnucleic acid single linear DNA 13 TCAGAGAAGT ACACCCATAA TCC 23 17 basepairs nucleic acid single linear DNA 14 AAGTACACCC ATAATCC 17 56 basepairs nucleic acid single unknown RNA 15 ACAGAAUUUU UGACAAAUCAAAGCAGANNN NUCUGAGNAG UCCUUACUUC AGAGAA 56 57 base pairs nucleic acidsingle unknown RNA 16 GGCCCGGGCA GCCUGCCCAA AGCCGGNNNN CCGGAGNAGUCGCCAGACCG GCUCGUG 57 56 base pairs nucleic acid single unknown RNA 17UGGCAUGCAA GACAAAGCAG GNNNNCCUGA GNAGUCCUUA AAUCUCCAAG GAGUAA 56 50 basepairs nucleic acid single unknown RNA 18 UAUAUGACAA AGCUGUNNNNACAGAGNAGU CCUUGUGUGG UAAAGACACG 50 61 base pairs nucleic acid singleunknown RNA 19 AGCACCAAUU GAAUUGAUGG CCAAAGCGGG NNNNCCCGAG NAGUCAACCGUAACAGUAUG 60 U 61 69 base pairs nucleic acid single unknown RNA 20UGAAAUUGUU UCAGGCUCCA AAGCCGGNNN NCCGGAGNAG UCAAGAAGAG GACCACAUGU 60CACUGAUGC 69 61 base pairs nucleic acid single unknown RNA 21 GGUUUCUUCAGUGAAAUUAC ACAAAGCAGC NNNNGCUGAG NAGUCAGUUA GGUCACACAU 60 C 61 53 basepairs nucleic acid single unknown RNA 22 ACCCAUUAUA ACACAAAGCUGANNNNUCAG AGNAGUCAUC UGAAGGUUUC UUC 53 17 base pairs nucleic acidsingle linear DNA 23 GCTGCACTTA ACCTGGC 17 16 base pairs nucleic acidsingle linear DNA 24 GGATAACTCA GGAACG 16 17 base pairs nucleic acidsingle linear DNA 25 CACTATTTGC CCTTCAG 17 17 base pairs nucleic acidsingle linear DNA 26 GCCTGAGATA GGGGTGC 17 17 base pairs nucleic acidsingle linear DNA 27 CACTATTTGC CCTTCAG 17 17 base pairs nucleic acidsingle linear DNA 28 GCCTGAGATA GGGGTGC 17 8 amino acids amino acidunknown peptide 29 Pro Asn Pro Lys Asn Cys Ser Trp 1 5 24 base pairsnucleic acid single linear DNA 30 CCAAACCCCA AGAATTGTTC CTGG 24 9 aminoacids amino acid unknown peptide 31 Lys Ile Met Glu Asn Lys Met Cys Asp1 5 26 base pairs nucleic acid single linear DNA 32 TCRCACATYTTRTTNCCCAT TATCTT 26 8 amino acids amino acid unknown peptide 33 Ala GlnGly Leu Asn Phe Gln Lys 1 5 24 base pairs nucleic acid single linear DNA34 GCACAAGGAC TGAATTTCCA AAAG 24 20 base pairs nucleic acid singlelinear DNA 35 CTGCCTGAAG TGTTAGAAGA 20 21 base pairs nucleic acid singlelinear DNA 36 GCTGAACTGA CATTAGAGGT G 21 25 base pairs nucleic acidsingle linear DNA 37 ACCTATGAGG ACGAAAGCCA GAGAC 25 24 base pairsnucleic acid single linear DNA 38 TGTGAGCAAC TGTCCTCGAG AACT 24 42 basepairs nucleic acid single linear DNA 39 GTCACGATGT CGACGTGTAC TTCTCTGAAGTAAGATGATT TG 42 53 base pairs nucleic acid single linear DNA 40GTCAGGTCAG AAAAGCTTAT CACTCTGTGT TTTTCAATAT CATCTTGAGT GAA 53 21 basepairs nucleic acid single linear DNA 41 AAGCTTTTCT GACCTGACNN N 21 3854base pairs nucleic acid double unknown cDNA Coding Sequence 61...3546 42GTCGACCCAC GCGTCCGGAG GAATCGTTCT GCAAATCCAG GTGTACACCT CTGAAGAAAG 60 ATGATG TGT CAG AAA TTC TAT GTG GTT TTG TTA CAC TGG GAA TTT CTT 108 Met MetCys Gln Lys Phe Tyr Val Val Leu Leu His Trp Glu Phe Leu 1 5 10 15 TATGTG ATA GCT GCA CTT AAC CTG GCA TAT CCA ATC TCT CCC TGG AAA 156 Tyr ValIle Ala Ala Leu Asn Leu Ala Tyr Pro Ile Ser Pro Trp Lys 20 25 30 TTT AAGTTG TTT TGT GGA CCA CCG AAC ACA ACC GAT GAC TCC TTT CTC 204 Phe Lys LeuPhe Cys Gly Pro Pro Asn Thr Thr Asp Asp Ser Phe Leu 35 40 45 TCA CCT GCTGGA GCC CCA AAC AAT GCC TCG GCT TTG AAG GGG GCT TCT 252 Ser Pro Ala GlyAla Pro Asn Asn Ala Ser Ala Leu Lys Gly Ala Ser 50 55 60 GAA GCA ATT GTTGAA GCT AAA TTT AAT TCA AGT GGT ATC TAC GTT CCT 300 Glu Ala Ile Val GluAla Lys Phe Asn Ser Ser Gly Ile Tyr Val Pro 65 70 75 80 GAG TTA TCC AAAACA GTC TTC CAC TGT TGC TTT GGG AAT GAG CAA GGT 348 Glu Leu Ser Lys ThrVal Phe His Cys Cys Phe Gly Asn Glu Gln Gly 85 90 95 CAA AAC TGC TCT GCACTC ACA GAC AAC ACT GAA GGG AAG ACA CTG GCT 396 Gln Asn Cys Ser Ala LeuThr Asp Asn Thr Glu Gly Lys Thr Leu Ala 100 105 110 TCA GTA GTG AAG GCTTCA GTT TTT CGC CAG CTA GGT GTA AAC TGG GAC 444 Ser Val Val Lys Ala SerVal Phe Arg Gln Leu Gly Val Asn Trp Asp 115 120 125 ATA GAG TGC TGG ATGAAA GGG GAC TTG ACA TTA TTC ATC TGT CAT ATG 492 Ile Glu Cys Trp Met LysGly Asp Leu Thr Leu Phe Ile Cys His Met 130 135 140 GAG CCA TTA CCT AAGAAC CCC TTC AAG AAT TAT GAC TCT AAG GTC CAT 540 Glu Pro Leu Pro Lys AsnPro Phe Lys Asn Tyr Asp Ser Lys Val His 145 150 155 160 CTT TTA TAT GATCTG CCT GAA GTC ATA GAT GAT TCG CCT CTG CCC CCA 588 Leu Leu Tyr Asp LeuPro Glu Val Ile Asp Asp Ser Pro Leu Pro Pro 165 170 175 CTG AAA GAC AGCTTT CAG ACT GTC CAA TGC AAC TGC AGT CTT CGG GGA 636 Leu Lys Asp Ser PheGln Thr Val Gln Cys Asn Cys Ser Leu Arg Gly 180 185 190 TGT GAA TGT CATGTG CCG GTA CCC AGA GCC AAA CTC AAC TAC GCT CTT 684 Cys Glu Cys His ValPro Val Pro Arg Ala Lys Leu Asn Tyr Ala Leu 195 200 205 CTG ATG TAT TTGGAA ATC ACA TCT GCC GGT GTG AGT TTT CAG TCA CCT 732 Leu Met Tyr Leu GluIle Thr Ser Ala Gly Val Ser Phe Gln Ser Pro 210 215 220 CTG ATG TCA CTGCAG CCC ATG CTT GTT GTG AAA CCC GAT CCA CCC TTA 780 Leu Met Ser Leu GlnPro Met Leu Val Val Lys Pro Asp Pro Pro Leu 225 230 235 240 GGT TTG CATATG GAA GTC ACA GAT GAT GGT AAT TTA AAG ATT TCT TGG 828 Gly Leu His MetGlu Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp 245 250 255 GAC AGC CAAACA ATG GCA CCA TTT CCG CTT CAA TAT CAG GTG AAA TAT 876 Asp Ser Gln ThrMet Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr 260 265 270 TTA GAG AATTCT ACA ATT GTA AGA GAG GCT GCT GAA ATT GTC TCA GCT 924 Leu Glu Asn SerThr Ile Val Arg Glu Ala Ala Glu Ile Val Ser Ala 275 280 285 ACA TCT CTGCTG GTA GAC AGT GTG CTT CCT GGA TCT TCA TAT GAG GTC 972 Thr Ser Leu LeuVal Asp Ser Val Leu Pro Gly Ser Ser Tyr Glu Val 290 295 300 CAG GTG AGGAGC AAG AGA CTG GAT GGT TCA GGA GTC TGG AGT GAC TGG 1020 Gln Val Arg SerLys Arg Leu Asp Gly Ser Gly Val Trp Ser Asp Trp 305 310 315 320 AGT TCACCT CAA GTC TTT ACC ACA CAA GAT GTT GTG TAT TTT CCA CCC 1068 Ser Ser ProGln Val Phe Thr Thr Gln Asp Val Val Tyr Phe Pro Pro 325 330 335 AAA ATTCTG ACT AGT GTT GGA TCG AAT GCT TCT TTT CAT TGC ATC TAC 1116 Lys Ile LeuThr Ser Val Gly Ser Asn Ala Ser Phe His Cys Ile Tyr 340 345 350 AAA AACGAA AAC CAG ATT ATC TCC TCA AAA CAG ATA GTT TGG TGG AGG 1164 Lys Asn GluAsn Gln Ile Ile Ser Ser Lys Gln Ile Val Trp Trp Arg 355 360 365 AAT CTAGCT GAG AAA ATC CCT GAG ATA CAG TAC AGC ATT GTG AGT GAC 1212 Asn Leu AlaGlu Lys Ile Pro Glu Ile Gln Tyr Ser Ile Val Ser Asp 370 375 380 CGA GTTAGC AAA GTT ACC TTC TCC AAC CTG AAA GCC ACC AGA CCT CGA 1260 Arg Val SerLys Val Thr Phe Ser Asn Leu Lys Ala Thr Arg Pro Arg 385 390 395 400 GGGAAG TTT ACC TAT GAC GCA GTG TAC TGC TGC AAT GAG CAG GCG TGC 1308 Gly LysPhe Thr Tyr Asp Ala Val Tyr Cys Cys Asn Glu Gln Ala Cys 405 410 415 CATCAC CGC TAT GCT GAA TTA TAC GTG ATC GAT GTC AAT ATC AAT ATA 1356 His HisArg Tyr Ala Glu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile 420 425 430 TCATGT GAA ACT GAC GGG TAC TTA ACT AAA ATG ACT TGC AGA TGG TCA 1404 Ser CysGlu Thr Asp Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser 435 440 445 CCCAGC ACA ATC CAA TCA CTA GTG GGA AGC ACT GTG CAG CTG AGG TAT 1452 Pro SerThr Ile Gln Ser Leu Val Gly Ser Thr Val Gln Leu Arg Tyr 450 455 460 CACAGG CGC AGC CTG TAT TGT CCT GAT AGT CCA TCT ATT CAT CCT ACG 1500 His ArgArg Ser Leu Tyr Cys Pro Asp Ser Pro Ser Ile His Pro Thr 465 470 475 480TCT GAG CCC AAA AAC TGC GTC TTA CAG AGA GAC GGC TTT TAT GAA TGT 1548 SerGlu Pro Lys Asn Cys Val Leu Gln Arg Asp Gly Phe Tyr Glu Cys 485 490 495GTT TTC CAG CCA ATC TTT CTA TTA TCT GGC TAT ACA ATG TGG ATC AGG 1596 ValPhe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg 500 505 510ATC AAC CAT TCT TTA GGT TCA CTT GAC TCG CCA CCA ACG TGT GTC CTT 1644 IleAsn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu 515 520 525CCT GAC TCC GTA GTA AAA CCA CTA CCT CCA TCT AAC GTA AAA GCA GAG 1692 ProAsp Ser Val Val Lys Pro Leu Pro Pro Ser Asn Val Lys Ala Glu 530 535 540ATT ACT GTA AAC ACT GGA TTA TTG AAA GTA TCT TGG GAA AAG CCA GTC 1740 IleThr Val Asn Thr Gly Leu Leu Lys Val Ser Trp Glu Lys Pro Val 545 550 555560 TTT CCG GAG AAT AAC CTT CAA TTC CAG ATT CGA TAT GGC TTA AGT GGA 1788Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg Tyr Gly Leu Ser Gly 565 570575 AAA GAA ATA CAA TGG AAG ACA CAT GAG GTA TTC GAT GCA AAG TCA AAG 1836Lys Glu Ile Gln Trp Lys Thr His Glu Val Phe Asp Ala Lys Ser Lys 580 585590 TCT GCC AGC CTG CTG GTG TCA GAC CTC TGT GCA GTC TAT GTG GTC CAG 1884Ser Ala Ser Leu Leu Val Ser Asp Leu Cys Ala Val Tyr Val Val Gln 595 600605 GTT CGC TGC CGG CGG TTG GAT GGA CTA GGA TAT TGG AGT AAT TGG AGC 1932Val Arg Cys Arg Arg Leu Asp Gly Leu Gly Tyr Trp Ser Asn Trp Ser 610 615620 AGT CCA GCC TAT ACG CTT GTC ATG GAT GTA AAA GTT CCT ATG AGA GGG 1980Ser Pro Ala Tyr Thr Leu Val Met Asp Val Lys Val Pro Met Arg Gly 625 630635 640 CCT GAA TTT TGG AGA AAA ATG GAT GGG GAC GTT ACT AAA AAG GAG AGA2028 Pro Glu Phe Trp Arg Lys Met Asp Gly Asp Val Thr Lys Lys Glu Arg 645650 655 AAT GTC ACC TTG CTT TGG AAG CCC CTG ACG AAA AAT GAC TCA CTG TGT2076 Asn Val Thr Leu Leu Trp Lys Pro Leu Thr Lys Asn Asp Ser Leu Cys 660665 670 AGT GTG AGG AGG TAC GTT GTG AAG CAT CGT ACT GCC CAC AAT GGG ACG2124 Ser Val Arg Arg Tyr Val Val Lys His Arg Thr Ala His Asn Gly Thr 675680 685 TGG TCA GAA GAT GTG GGA AAT CGG ACC AAT CTC ACT TTC CTG TGG ACA2172 Trp Ser Glu Asp Val Gly Asn Arg Thr Asn Leu Thr Phe Leu Trp Thr 690695 700 GAA CCA GCG CAC ACT GTT ACA GTT CTG GCT GTC AAT TCC CTC GGC GCT2220 Glu Pro Ala His Thr Val Thr Val Leu Ala Val Asn Ser Leu Gly Ala 705710 715 720 TCC CTT GTG AAT TTT AAC CTT ACC TTC TCA TGG CCC ATG AGT AAAGTG 2268 Ser Leu Val Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val725 730 735 AGT GCT GTG GAG TCA CTC AGT GCT TAT CCC CTG AGC AGC AGC TGTGTC 2316 Ser Ala Val Glu Ser Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val740 745 750 ATC CTT TCC TGG ACA CTG TCA CCT GAT GAT TAT AGT CTG TTA TATCTG 2364 Ile Leu Ser Trp Thr Leu Ser Pro Asp Asp Tyr Ser Leu Leu Tyr Leu755 760 765 GTT ATT GAA TGG AAG ATC CTT AAT GAA GAT GAT GGA ATG AAG TGGCTT 2412 Val Ile Glu Trp Lys Ile Leu Asn Glu Asp Asp Gly Met Lys Trp Leu770 775 780 AGA ATT CCC TCG AAT GTT AAA AAG TTT TAT ATC CAC GAT AAT TTTATT 2460 Arg Ile Pro Ser Asn Val Lys Lys Phe Tyr Ile His Asp Asn Phe Ile785 790 795 800 CCC ATC GAG AAA TAT CAG TTT AGT CTT TAC CCA GTA TTT ATGGAA GGA 2508 Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro Val Phe Met GluGly 805 810 815 GTT GGA AAA CCA AAG ATA ATT AAT GGT TTC ACC AAA GAT GCTATC GAC 2556 Val Gly Lys Pro Lys Ile Ile Asn Gly Phe Thr Lys Asp Ala IleAsp 820 825 830 AAG CAG CAG AAT GAC GCA GGG CTG TAT GTC ATT GTA CCC ATAATT ATT 2604 Lys Gln Gln Asn Asp Ala Gly Leu Tyr Val Ile Val Pro Ile IleIle 835 840 845 TCC TCT TGT GTC CTA CTG CTC GGA ACA CTG TTA ATT TCA CACCAG AGA 2652 Ser Ser Cys Val Leu Leu Leu Gly Thr Leu Leu Ile Ser His GlnArg 850 855 860 ATG AAA AAG TTG TTT TGG GAC GAT GTT CCA AAC CCC AAG AATTGT TCC 2700 Met Lys Lys Leu Phe Trp Asp Asp Val Pro Asn Pro Lys Asn CysSer 865 870 875 880 TGG GCA CAA GGA CTG AAT TTC CAA AAG CCT GAA ACA TTTGAG CAT CTT 2748 Trp Ala Gln Gly Leu Asn Phe Gln Lys Pro Glu Thr Phe GluHis Leu 885 890 895 TTT ACC AAG CAT GCA GAA TCA GTG ATA TTT GGT CCT CTTCTT CTG GAG 2796 Phe Thr Lys His Ala Glu Ser Val Ile Phe Gly Pro Leu LeuLeu Glu 900 905 910 CCT GAA CCC ATT TCA GAA GAA ATC AGT GTC GAT ACA GCTTGG AAA AAT 2844 Pro Glu Pro Ile Ser Glu Glu Ile Ser Val Asp Thr Ala TrpLys Asn 915 920 925 AAA GAT GAG ATG GTC CCA GCA GCT ATG GTC TCC CTT CTTTTG ACC ACA 2892 Lys Asp Glu Met Val Pro Ala Ala Met Val Ser Leu Leu LeuThr Thr 930 935 940 CCA GAC CCT GAA AGC AGT TCT ATT TGT ATT AGT GAC CAGTGT AAC AGT 2940 Pro Asp Pro Glu Ser Ser Ser Ile Cys Ile Ser Asp Gln CysAsn Ser 945 950 955 960 GCT AAC TTC TCT GGG TCT CAG AGC ACC CAG GTA ACCTGT GAG GAT GAG 2988 Ala Asn Phe Ser Gly Ser Gln Ser Thr Gln Val Thr CysGlu Asp Glu 965 970 975 TGT CAG AGA CAA CCC TCA GTT AAA TAT GCA ACT CTGGTC AGC AAC GAT 3036 Cys Gln Arg Gln Pro Ser Val Lys Tyr Ala Thr Leu ValSer Asn Asp 980 985 990 AAA CTA GTG GAA ACT GAT GAA GAG CAA GGG TTT ATCCAT AGT CCT GTC 3084 Lys Leu Val Glu Thr Asp Glu Glu Gln Gly Phe Ile HisSer Pro Val 995 1000 1005 AGC AAC TGC ATC TCC AGT AAT CAT TCC CCA CTGAGG CAG TCT TTC TCT 3132 Ser Asn Cys Ile Ser Ser Asn His Ser Pro Leu ArgGln Ser Phe Ser 1010 1015 1020 AGC AGC TCC TGG GAG ACA GAG GCC CAG ACATTT TTC CTT TTA TCA GAC 3180 Ser Ser Ser Trp Glu Thr Glu Ala Gln Thr PhePhe Leu Leu Ser Asp 1025 1030 1035 1040 CAG CAA CCC ACC ATG ATT TCA CCACAA CTT TCA TTC TCG GGG TTG GAT 3228 Gln Gln Pro Thr Met Ile Ser Pro GlnLeu Ser Phe Ser Gly Leu Asp 1045 1050 1055 GAG CTT TTG GAA CTG GAG GGAAGT TTT CCT GAA GAA AAT CAC AGG GAG 3276 Glu Leu Leu Glu Leu Glu Gly SerPhe Pro Glu Glu Asn His Arg Glu 1060 1065 1070 AAG TCT GTC TGT TAT CTAGGA GTC ACC TCC GTC AAC AGA AGA GAG AGT 3324 Lys Ser Val Cys Tyr Leu GlyVal Thr Ser Val Asn Arg Arg Glu Ser 1075 1080 1085 GGT GTG CTT TTG ACTGGT GAG GCA GGA ATC CTG TGC ACA TTC CCA GCC 3372 Gly Val Leu Leu Thr GlyGlu Ala Gly Ile Leu Cys Thr Phe Pro Ala 1090 1095 1100 CAG TGT CTG TTCACT GAC ATC AGG ATC CTC CAG GAG AGA TGC TCA CAC 3420 Gln Cys Leu Phe ThrAsp Ile Arg Ile Leu Gln Glu Arg Cys Ser His 1105 1110 1115 1120 TTT GTAGAA AAT AAT TTG AGT TTA GGG ACC TCT GGT GAG AAC TTT GTA 3468 Phe Val GluAsn Asn Leu Ser Leu Gly Thr Ser Gly Glu Asn Phe Val 1125 1130 1135 CCTTAC ATG CCC CAA TTT CAA ACC TGT TCC ACG CAC AGT CAC AAG ATA 3516 Pro TyrMet Pro Gln Phe Gln Thr Cys Ser Thr His Ser His Lys Ile 1140 1145 1150ATG GAG AAT AAG ATG TGT GAC TTA ACT GTG TAATCTCATC CAAGAAGCCT 3566 MetGlu Asn Lys Met Cys Asp Leu Thr Val 1155 1160 CAAGGTTCCA TTCCAGTAGAGCCTGTCATG TATAATGTGT TCTTTTATTG TTGTGGATGT 3626 GGGAGACAAG TGTCAGAATCTAGTGTGAAA ATGATTGTTT CCAAACTAAG TGTGTCTATT 3686 TTCTCTCAGT AATACANATGAAACATATGA GGAAGCCCTC ATTAATCTAC TAATGTAGAT 3746 GGACTCTTAC TGAATATATTCCCAAGATAC TTGGGGAAGT CTCCCTAATT CTAGCTAAAA 3806 GAANTAGAAC TACTAAACACTGAATCTGGA AAAAAAAAAA AAAAAAAG 3854 1162 amino acids amino acid unknownprotein internal 43 Met Met Cys Gln Lys Phe Tyr Val Val Leu Leu His TrpGlu Phe Leu 1 5 10 15 Tyr Val Ile Ala Ala Leu Asn Leu Ala Tyr Pro IleSer Pro Trp Lys 20 25 30 Phe Lys Leu Phe Cys Gly Pro Pro Asn Thr Thr AspAsp Ser Phe Leu 35 40 45 Ser Pro Ala Gly Ala Pro Asn Asn Ala Ser Ala LeuLys Gly Ala Ser 50 55 60 Glu Ala Ile Val Glu Ala Lys Phe Asn Ser Ser GlyIle Tyr Val Pro 65 70 75 80 Glu Leu Ser Lys Thr Val Phe His Cys Cys PheGly Asn Glu Gln Gly 85 90 95 Gln Asn Cys Ser Ala Leu Thr Asp Asn Thr GluGly Lys Thr Leu Ala 100 105 110 Ser Val Val Lys Ala Ser Val Phe Arg GlnLeu Gly Val Asn Trp Asp 115 120 125 Ile Glu Cys Trp Met Lys Gly Asp LeuThr Leu Phe Ile Cys His Met 130 135 140 Glu Pro Leu Pro Lys Asn Pro PheLys Asn Tyr Asp Ser Lys Val His 145 150 155 160 Leu Leu Tyr Asp Leu ProGlu Val Ile Asp Asp Ser Pro Leu Pro Pro 165 170 175 Leu Lys Asp Ser PheGln Thr Val Gln Cys Asn Cys Ser Leu Arg Gly 180 185 190 Cys Glu Cys HisVal Pro Val Pro Arg Ala Lys Leu Asn Tyr Ala Leu 195 200 205 Leu Met TyrLeu Glu Ile Thr Ser Ala Gly Val Ser Phe Gln Ser Pro 210 215 220 Leu MetSer Leu Gln Pro Met Leu Val Val Lys Pro Asp Pro Pro Leu 225 230 235 240Gly Leu His Met Glu Val Thr Asp Asp Gly Asn Leu Lys Ile Ser Trp 245 250255 Asp Ser Gln Thr Met Ala Pro Phe Pro Leu Gln Tyr Gln Val Lys Tyr 260265 270 Leu Glu Asn Ser Thr Ile Val Arg Glu Ala Ala Glu Ile Val Ser Ala275 280 285 Thr Ser Leu Leu Val Asp Ser Val Leu Pro Gly Ser Ser Tyr GluVal 290 295 300 Gln Val Arg Ser Lys Arg Leu Asp Gly Ser Gly Val Trp SerAsp Trp 305 310 315 320 Ser Ser Pro Gln Val Phe Thr Thr Gln Asp Val ValTyr Phe Pro Pro 325 330 335 Lys Ile Leu Thr Ser Val Gly Ser Asn Ala SerPhe His Cys Ile Tyr 340 345 350 Lys Asn Glu Asn Gln Ile Ile Ser Ser LysGln Ile Val Trp Trp Arg 355 360 365 Asn Leu Ala Glu Lys Ile Pro Glu IleGln Tyr Ser Ile Val Ser Asp 370 375 380 Arg Val Ser Lys Val Thr Phe SerAsn Leu Lys Ala Thr Arg Pro Arg 385 390 395 400 Gly Lys Phe Thr Tyr AspAla Val Tyr Cys Cys Asn Glu Gln Ala Cys 405 410 415 His His Arg Tyr AlaGlu Leu Tyr Val Ile Asp Val Asn Ile Asn Ile 420 425 430 Ser Cys Glu ThrAsp Gly Tyr Leu Thr Lys Met Thr Cys Arg Trp Ser 435 440 445 Pro Ser ThrIle Gln Ser Leu Val Gly Ser Thr Val Gln Leu Arg Tyr 450 455 460 His ArgArg Ser Leu Tyr Cys Pro Asp Ser Pro Ser Ile His Pro Thr 465 470 475 480Ser Glu Pro Lys Asn Cys Val Leu Gln Arg Asp Gly Phe Tyr Glu Cys 485 490495 Val Phe Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp Ile Arg 500505 510 Ile Asn His Ser Leu Gly Ser Leu Asp Ser Pro Pro Thr Cys Val Leu515 520 525 Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Asn Val Lys AlaGlu 530 535 540 Ile Thr Val Asn Thr Gly Leu Leu Lys Val Ser Trp Glu LysPro Val 545 550 555 560 Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg TyrGly Leu Ser Gly 565 570 575 Lys Glu Ile Gln Trp Lys Thr His Glu Val PheAsp Ala Lys Ser Lys 580 585 590 Ser Ala Ser Leu Leu Val Ser Asp Leu CysAla Val Tyr Val Val Gln 595 600 605 Val Arg Cys Arg Arg Leu Asp Gly LeuGly Tyr Trp Ser Asn Trp Ser 610 615 620 Ser Pro Ala Tyr Thr Leu Val MetAsp Val Lys Val Pro Met Arg Gly 625 630 635 640 Pro Glu Phe Trp Arg LysMet Asp Gly Asp Val Thr Lys Lys Glu Arg 645 650 655 Asn Val Thr Leu LeuTrp Lys Pro Leu Thr Lys Asn Asp Ser Leu Cys 660 665 670 Ser Val Arg ArgTyr Val Val Lys His Arg Thr Ala His Asn Gly Thr 675 680 685 Trp Ser GluAsp Val Gly Asn Arg Thr Asn Leu Thr Phe Leu Trp Thr 690 695 700 Glu ProAla His Thr Val Thr Val Leu Ala Val Asn Ser Leu Gly Ala 705 710 715 720Ser Leu Val Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser Lys Val 725 730735 Ser Ala Val Glu Ser Leu Ser Ala Tyr Pro Leu Ser Ser Ser Cys Val 740745 750 Ile Leu Ser Trp Thr Leu Ser Pro Asp Asp Tyr Ser Leu Leu Tyr Leu755 760 765 Val Ile Glu Trp Lys Ile Leu Asn Glu Asp Asp Gly Met Lys TrpLeu 770 775 780 Arg Ile Pro Ser Asn Val Lys Lys Phe Tyr Ile His Asp AsnPhe Ile 785 790 795 800 Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro ValPhe Met Glu Gly 805 810 815 Val Gly Lys Pro Lys Ile Ile Asn Gly Phe ThrLys Asp Ala Ile Asp 820 825 830 Lys Gln Gln Asn Asp Ala Gly Leu Tyr ValIle Val Pro Ile Ile Ile 835 840 845 Ser Ser Cys Val Leu Leu Leu Gly ThrLeu Leu Ile Ser His Gln Arg 850 855 860 Met Lys Lys Leu Phe Trp Asp AspVal Pro Asn Pro Lys Asn Cys Ser 865 870 875 880 Trp Ala Gln Gly Leu AsnPhe Gln Lys Pro Glu Thr Phe Glu His Leu 885 890 895 Phe Thr Lys His AlaGlu Ser Val Ile Phe Gly Pro Leu Leu Leu Glu 900 905 910 Pro Glu Pro IleSer Glu Glu Ile Ser Val Asp Thr Ala Trp Lys Asn 915 920 925 Lys Asp GluMet Val Pro Ala Ala Met Val Ser Leu Leu Leu Thr Thr 930 935 940 Pro AspPro Glu Ser Ser Ser Ile Cys Ile Ser Asp Gln Cys Asn Ser 945 950 955 960Ala Asn Phe Ser Gly Ser Gln Ser Thr Gln Val Thr Cys Glu Asp Glu 965 970975 Cys Gln Arg Gln Pro Ser Val Lys Tyr Ala Thr Leu Val Ser Asn Asp 980985 990 Lys Leu Val Glu Thr Asp Glu Glu Gln Gly Phe Ile His Ser Pro Val995 1000 1005 Ser Asn Cys Ile Ser Ser Asn His Ser Pro Leu Arg Gln SerPhe Ser 1010 1015 1020 Ser Ser Ser Trp Glu Thr Glu Ala Gln Thr Phe PheLeu Leu Ser Asp 025 1030 1035 1040 Gln Gln Pro Thr Met Ile Ser Pro GlnLeu Ser Phe Ser Gly Leu Asp 1045 1050 1055 Glu Leu Leu Glu Leu Glu GlySer Phe Pro Glu Glu Asn His Arg Glu 1060 1065 1070 Lys Ser Val Cys TyrLeu Gly Val Thr Ser Val Asn Arg Arg Glu Ser 1075 1080 1085 Gly Val LeuLeu Thr Gly Glu Ala Gly Ile Leu Cys Thr Phe Pro Ala 1090 1095 1100 GlnCys Leu Phe Thr Asp Ile Arg Ile Leu Gln Glu Arg Cys Ser His 105 11101115 1120 Phe Val Glu Asn Asn Leu Ser Leu Gly Thr Ser Gly Glu Asn PheVal 1125 1130 1135 Pro Tyr Met Pro Gln Phe Gln Thr Cys Ser Thr His SerHis Lys Ile 1140 1145 1150 Met Glu Asn Lys Met Cys Asp Leu Thr Val 11551160 26 base pairs nucleic acid double unknown cDNA 44 TCRCACATYTTRTTNCCCAT TATCTT 26 33 base pairs nucleic acid single linear DNA 45CCCAATGTCG ACATGATGTG TCAGAAATTC TAT 33 33 base pairs nucleic acidsingle linear DNA 46 AAAAAGGATC CGGTCATTCT GCTGCTTGTC GAT 33 33 basepairs nucleic acid single linear DNA 47 CCCAATGTCG ACATGGTGTA CTTCTCTGAAGTA 33 30 base pairs nucleic acid single linear DNA 48 TTTTTGGATCCCACCTGCAT CACTCTGGTG 30 48 base pairs nucleic acid single linear DNA 49TTTAACTTGT CATATCCAAT TACTCCTTGG AGATTTAAGT TGTCTTGC 48 30 base pairsnucleic acid single linear DNA 50 TTTTTGGATC CCACCTGCAT CACTCTGGTG 30

What is claimed is:
 1. A chimeric protein comprising amino acids 23-1162of SEQ ID NO:43 covalently attached by a peptide bond to a constantregion of an immunoglobulin.
 2. The chimeric protein of claim 1consisting essentially of amino acids 23-1162 of SEQ ID NO:43 covalentlyattached by a peptide bond to a constant region of an immunoglobulin. 3.A chimeric protein comprising amino acids 23-894 of SEQ ID NO;2covalently attached by a peptide bond to a constant region of animmunoglobulin.
 4. A chimeric protein of claim 3 consisting essentiallyof amino acids 23-894 of SEQ ID NO:2 covalently attached by a peptidebond to a constant region of an immunoglobulin.
 5. A chimeric proteinconsisting of amino acids 23-1162 of SEQ ID NO:43 covalently attached bya peptide bond to a constant region of an immunoglobulin.
 6. A chimericprotein consisting of amino acids 23-894 of SEQ ID NO:2 covalentlyattached by a peptide bond to a constant region of an immunoglobulin. 7.A chimeric protein comprising amino acids 1-1162 of SEQ ID NO:43covalently attached by a peptide bond to a constant region of animmunoglobulin.
 8. The chimeric protein of claim 7 consistingessentially of amino acids 1-1162 of SEQ ID NO:43 covalently attached bya peptide bond to a constant region of an immunoglobulin.
 9. A chimericprotein comprising amino acids 1-894 of SEQ ID NO:2 covalently attachedby a peptide bond to a constant region of an immunoglobulin.
 10. Achimeric protein of claim 9 consisting essentially of amino acids 1-894of SEQ ID NO:2 covalently attached by a peptide bond to a constantregion of an immunoglobulin.
 11. A chimeric protein consisting of aminoacids 1-1162 of SEQ ID NO:43 covalently attached by a peptide bond to aconstant region of an immunoglobulin.
 12. A chimeric protein consistingof amino acids 1-894 of SEQ ID NO:2 covalently attached by a peptidebond to a constant region of an immunoglobulin.
 13. A chimeric proteincomprising amino acids 23-837 of SEQ ID NO:43 covalently attached by apeptide bond to a constant region of an immunoglobutin.
 14. The chimericprotein of claim 13 consisting essentially of amino acids 23-837 of SEQID NO:43 covalently attached by a peptide bond to a constant region ofan immunoglobulin.
 15. A chimeric protein comprising amino acids 23-860of SEQ ID NO:43 covalently attachedby a peptide bond to a constantregion of an immunoglobulin.
 16. A chimeric protein of claim 15consisting essentially of amino acids 23-860 of SEQ ID NO:43 covalentlyattached by a peptide bond to a constant region of an immunoglobulin.17. A chimeric protein consisting of amino acids 23-837 of SEQ ID NO:43covalently attached by a peptide bond to a constant region of animmunoglobulin.
 18. A chimeric protein consisting of amino acids 23-860of SEQ ID NO:43 covalently attached by a peptide bond to a constantregion of an immunoglobulin.
 19. A chimeric protein comprising aminoacids 1-837 of SEQ ID NO:43 covalently attached by a peptide bond to aconstant region of an immunoglobulin.
 20. The chimeric protein of claim19 consisting essentially of amino acids 1-837 of SEQ ID NO:43covalently attached by a peptide bond to a constant region of animmunoglobulin.
 21. A chimeric protein comprising amino acids 1-860 ofSEQ ID NO:43 covalently attached by a peptide bond to a constant regionof an immunoglobulin.
 22. A chimeric protein of claim 21 consistingessentially of amino acids 1-860 of SEQ ID NO:43 covalently attached bya peptide bond to a constant region of an immunoglobulin.
 23. A chimericprotein consisting of amino acids 1-837 of SEQ ID NO:43 covalentlyattached by a peptide bond to a constant region of an immunoglobulin.24. A chimeric protein consisting of amino acids 1-860 of SEQ ID NO:43covalently attached by a peptide bond to a constant region of animmunoglobulin.